De Soto sailed to the New World with Pedrarias Dávila, appointed as the first Governor of Panama. In 1520 he participated in Gaspar de Espinosa’s expedition to Veragua, and in 1524, he participated in the conquest of Nicaragua under Francisco Hernández de Córdoba. There he acquired an encomienda and a public office in León, Nicaragua. Brave leadership, unwavering loyalty, and ruthless schemes for the extortion of native villages for their captured chiefs became de Soto’s hallmarks during the conquest of Central America. He gained fame as an excellent horseman, fighter, and tactician. During that time, de Soto was influenced by the achievements of Iberian explorers: Juan Ponce de León, the first European to reach Florida; Vasco Núñez de Balboa, the first European to reach the Pacific Ocean coast of the Americas (he called it the “South Sea” on the south coast of Panama); and Ferdinand Magellan, who first sailed that ocean to East Asia. In 1530, de Soto became a regidor of León, Nicaragua. He led an expedition up the coast of the Yucatán Peninsula searching for a passage between the Atlantic Ocean and the Pacific Ocean to enable trade with the Orient, the richest market in the world. Failing that, and without means to explore further, de Soto, upon Pedro Arias Dávila’s death, left his estates in Nicaragua. Bringing his own men on ships which he hired, de Soto joined Francisco Pizarro at his first base of Tumbes shortly before departure for the interior of present-day Peru.

During 1533, the Spanish held Atahualpa captive in Cajamarca for months while his subjects paid for his ransom by filling a room with gold and silver objects. During this captivity, de Soto became friendly with Atahualpa and taught him to play chess. By the time the ransom had been completed, the Spanish became alarmed by rumors of an Inca army advancing on Cajamarca. Pizarro sent de Soto with 200 soldiers to scout for the rumored army.

By 1534, de Soto was serving as lieutenant governor of Cuzco while Pizarro was building his new capital on the coast; it later became known as Lima. In 1535 King Charles awarded Diego de Almagro, Francisco Pizarro’s partner, the governorship of the southern portion of the Inca Empire. When de Almagro made plans to explore and conquer the southern part of the Inca empire (now Chile), de Soto applied to be his second-in-command, but de Almagro turned him down. De Soto packed up his treasure and returned to Spain.

De Soto returned to Spain in 1536, with wealth gathered from plunder in the Spanish conquest of the Inca Empire. He was admitted into the prestigious Order of Santiago and “granted the right to conquer Florida”. His share was awarded to him by the King of Spain, and he received 724 marks of gold, and 17,740 pesos. He married Isabel de Bobadilla, daughter of Pedrarias Dávila and a relative of a confidante of Queen Isabella.

De Soto wrote a new will before embarking on his travels. On 10 May 1539, he wrote in his will:

In May 1539, de Soto landed nine ships with over 620 men and 220 horses in an area generally identified as south Tampa Bay. Historian Robert S. Weddle has suggested that he landed at either Charlotte Harbor or San Carlos Bay. He named the land as Espíritu Santo after the Holy Spirit. The ships carried priests, craftsmen, engineers, farmers, and merchants; some with their families, some from Cuba, most from Europe and Africa. Few of the men had traveled before outside of Spain, or even away from their home villages.

Ortiz developed a method for guiding the expedition and communicating with the various tribes, who spoke many dialects and languages. He recruited guides from each tribe along the route. A chain of communication was established whereby a guide who had lived in close proximity to another tribal area was able to pass his information and language on to a guide from a neighboring area. Because Ortiz refused to dress as an hidalgo Spaniard, other officers questioned his motives. De Soto remained loyal to Ortiz, allowing him the freedom to dress and live among his native friends. Another important guide was the seventeen-year-old boy Perico, or Pedro, from what is now Georgia. He spoke several of the local tribes’ languages and could communicate with Ortiz. Perico was taken as a guide in 1540. The Spanish had also captured other Indians, whom they used as slave labor. Perico was treated better due to his value to the Spaniards.

On May 8, 1541, de Soto’s troops reached the Mississippi River.

After a harsh winter, the Spanish expedition decamped and moved on more erratically. Their interpreter Juan Ortiz had died, making it more difficult for them to get directions and food sources, and generally to communicate with the Natives. The expedition went as far inland as the Caddo River, where they clashed with a Native American tribe called the Tula in October 1541. The Spaniards characterized them as the most skilled and dangerous warriors they had encountered. This may have happened in the area of present-day Caddo Gap, Arkansas (a monument to the de Soto expedition was erected in that community). Eventually, the Spaniards returned to the Mississippi River.

De Soto died of a fever on May 21, 1542, in the native village of Guachoya (historical sources disagree as to whether de Soto died near present-day McArthur, Arkansas, or in Louisiana) on the western bank of the Mississippi. Louisiana erected a historical marker at the estimated site.

De Soto had little interest in the river, which in his view was an obstacle to his mission. There has been considerable research into the exact location where de Soto crossed the Mississippi River. A commission appointed by Franklin D. Roosevelt in 1935 determined that Sunflower Landing, Mississippi, was the “most likely” crossing place. De Soto and his men spent a month building flatboats, and crossed the river at night to avoid the Native Americans who were patrolling the river. De Soto had hostile relations with the native people in this area.

Historians have worked to trace the route of de Soto’s expedition in North America, a controversial process over the years. Local politicians vied to have their localities associated with the expedition. The most widely used version of “De Soto’s Trail” comes from a study commissioned by the United States Congress. A committee chaired by the anthropologist John R. Swanton published The Final Report of the United States De Soto Expedition Commission in 1939. Among other locations, Manatee County, Florida, claims an approximate landing site for de Soto and has a national memorial recognizing that event. In the early 21st century, the first part of the expedition’s course, up to de Soto’s battle at Mabila (a small fortress town in present-day central Alabama), is disputed only in minor details. His route beyond Mabila is contested. Swanton reported the de Soto trail ran from there through Mississippi, Arkansas, and Texas.

De Soto headed north into the Appalachian Mountains of present-day western North Carolina, where he spent a month resting the horses while his men searched for gold. De Soto next entered eastern Tennessee. At this point, De Soto either continued along the Tennessee River to enter Alabama from the north (according to John R. Swanton), or turned south and entered northern Georgia (according to Charles M. Hudson). The route that Swanton proposed in 1939 is still generally accepted by most archaeologists and by the U.S. government as the route of the de Soto expedition..

The Governor Martin Site at the former Apalachee village of Anhaica, located about a mile east of the present Florida capital in Tallahassee, has been documented as definitively associated with de Soto’s expedition. The Governor Martin Site was discovered by archaeologist B. Calvin Jones in March 1987. It has been preserved as the DeSoto Site Historic State Park.

From their winter location in the western panhandle of Florida, having heard of gold being mined “toward the sun’s rising”, the expedition turned northeast through what is now the modern state of Georgia. Based on archaeological finds made in 2009 at a remote, privately owned site near the Ocmulgee River, researchers believe that de Soto’s expedition stopped in Telfair County. Artifacts found here include nine glass trade beads, some of which bear a chevron pattern made in Venice for a limited period of time and believed to be indicative of the de Soto expedition. Six metal objects were also found, including a silver pendant and some iron tools. The rarest items were found within what researchers believe was a large council house of the indigenous people whom de Soto was visiting.

As of 2016, the Richardson/UF Village site (8AL100) in Alachua County, west of Orange Lake, appears to have been accepted by archaeologists as the site of the town of Potano visited by the de Soto expedition. The 17th-century mission of San Buenaventura de Potano is believed to have been founded here.