Williamson was born in Madison County, Tennessee, near Jackson, in 1914. His original recordings are in the country blues style, but he soon demonstrated skill at making the harmonica a lead instrument for the blues and popularized it for the first time in a more urban blues setting. He has been called “the father of modern blues harp”. While in his teens he joined Yank Rachell and Sleepy John Estes, playing with them in Tennessee and Arkansas. In 1934 he settled in Chicago.

Williamson first recorded in 1937, for Bluebird Records, and his first recording, “Good Morning, School Girl”, became a standard. He was popular among black audiences throughout the southern United States and in Midwestern industrial cities, such as Detroit and Chicago, and his name was synonymous with the blues harmonica for the next decade. Other well-known recordings of his include “Sugar Mama Blues”, “Shake the Boogie”, “You Better Cut That Out”, “Sloppy Drunk”, “Early in the Morning”, “Stop Breaking Down”, and “Hoodoo Hoodoo” (also known as “Hoodoo Man Blues”). In 1947, “Shake the Boogie” made number 4 on Billboard’s Race Records chart. Williamson’s style influenced many blues harmonica performers, including Billy Boy Arnold, Junior Wells, Sonny Terry, Little Walter, and Snooky Pryor. He was the most widely heard and influential blues harmonica player of his generation. His music was also influential on many of his non-harmonica-playing contemporaries and successors, including Muddy Waters (who played guitar with Williamson in the mid-1940s) and Jimmy Rogers (whose first recording in 1946 was as a harmonica player, performing an uncanny imitation of Williamson’s style). These and other artists, both blues and rock, have helped popularize his songs through subsequent recordings.

Williamson’s final recording session took place in Chicago in December 1947, in which he accompanied Big Joe Williams. On June 1, 1948, Williamson was killed in a robbery on Chicago’s South Side as he walked home from a performance at the Plantation Club, at 31st St. and Giles Avenue, a tavern just a block and a half from his home, at 3226 S. Giles. Williamson’s final words are reported to have been “Lord have mercy”.

To add to the confusion, around 1940 the jazz pianist and singer Enoch Williams recorded for Decca under the name Sonny Boy Williams and in 1947 as Sunny Boy in the Sunny Boy Trio.

His legacy has been somewhat overshadowed in the postwar blues era by the popularity of the musician who appropriated his name, Rice Miller, who after Williamson’s death went on to record many popular blues songs for Chicago’s Checker Records and others and toured Europe several times during the blues revival in the 1960s. The recordings made by Williamson between 1937 and his death in 1948 and those made later by Rice Miller were all originally issued under the name Sonny Boy Williamson. It is believed that Miller adopted the name to deceive audiences (and his first record label) into thinking that he was the “original” Sonny Boy. In order to differentiate between the two musicians, many later scholars and biographers have referred to John Lee Williamson (1914–1948) as Sonny Boy Williamson I and Miller (c. 1912–1965) as Sonny Boy Williamson II.

Williamson is buried at the former site of the Blairs Chapel Church, southwest of Jackson, Tennessee. In 1991, a red granite marker was purchased by fans and family to mark the site of his burial. A Tennessee historical marker, also placed in 1991, indicates the place of his birth and describes his influence on blues music. The historical marker is located south of Jackson on Tennessee State Highway 18, at the corner of Caldwell Road.

Specialty labels, such as JSP Records, Saga, Indigo, Snapper, and others, have also released compilations. In 1991, Document Records issued Williamson’s Complete Recorded Works In Chronological Order as five CDs.