Playing for Scranton/Wilkes-Barre in 2008, in 94 games Gardner was second in the International League with a .414 on-base percentage, 70 walks, 11 triples, and was 6th in the IL with 37 stolen bases, while being caught only nine times.
Through 2008 in the minor leagues, he had a .291 batting average and a .389 OBP. He had stolen 153 bases, being caught only 31 times (an 83% success rate).
On June 30, 2008, Gardner was called up and made his major league debut, batting leadoff and going 0-for-3 with a stolen base. On July 2, he earned both his first hit and first RBI, off fellow rookie Texas Rangers relief pitcher Warner Madrigal in the seventh inning. Gardner went on to steal second and eventually score in that inning. On July 26, 2008, Gardner was optioned back to AAA after the acquisition of Xavier Nady in order to continue to receive playing time. The Yankees again recalled Gardner on August 15.
On September 21, 2008, Gardner scored the final run of Major League Baseball in Yankee Stadium history as a pinch runner for Jason Giambi, scoring on a sacrifice fly by Robinson Canó in the seventh inning of an eventual 7–3 win for the Yankees over the Baltimore Orioles. Gardner finished his rookie season playing 42 games, batting .228 with 16 RBI and 13 stolen bases.