Born in Centerville, Texas, Hopkins learned the blues when young in Buffalo, Texas from Blind Lemon Jefferson and his older cousin, country-blues singer Alger 'Texas' Alexander. When Hopkins and Alexander were playing in Houston in 1946, he was discovered by Lola Anne Cullum of Los Angeles' Aladdin Records (although Alexander would not make it out to L.A.) Hopkins' fast finger style is very distinct.

He settled in Houston in 1952 and gained much attention. Solid recordings followed including his masterpiece song Mojo Hand in 1960.

His style was born from spending many hours playing informally without a backing band. His distinctive style often included playing, in effect, bass, rhythm, lead, percussion, and vocals, all at the same time. His musical phrasing would often include a long low note at the beginning, the rhythm played in the middle range, then the lead in the high range. By playing this quickly - with occasional slaps of the guitar - the effect of bass, rhythm, percussion and lead would be created.

In 1968 Hopkins recorded the album Free Form Patterns backed by psychedelic rock band the 13th Floor Elevators.

Influence
Hopkins was a great influence on many local musicians around Houston and Austin, Texas in the 1950s and 1960s. He was an influence on Jimmie Vaughan's work and on the vocals and blues style of Ron "Pigpen" McKernan, the keyboardist of the Grateful Dead until 1972. He was also an important influence on Townes Van Zandt, the Texan folk/blues songwriter and performer, who often performed Hopkins numbers in his live performances. Doyle Bramhall II is another Texas artist who was influenced by Hopkins, as evidenced by a tattoo of Lightning on his upper left arm. Jimi Hendrix reportedly became interested in blues music listening to Lightnin' Hopkins records with his father.

The Houston Chronicle included Hopkins in their list of "100 Tall Texans", 100 important Texans that influenced the world. The George Bush Presidential Library and Museum included Hopkins in a 100 Tall Texans exhibit that opened in September 2006. The display includes Lightnin's Guild Starfire electric guitar and performance video.

Hopkins' Gibson J-160e guitar is on display at the Rock n' Roll Hall of Fame in Cleveland, Ohio.