Cavalli-Sforza, who was born in Genoa, Italy, was educated at the University of Pavia where he gained his MD in 1944. After working on bacterial genetics at Cambridge (1948–50) and Milan (1950–57) he held chairs in genetics at Parma (1958–62) and Pavia (1962–70). In 1970 he was appointed professor of genetics at the University of Stanford, California, a position he held until his retirement in 1992.

Cavalli-Sforza specialized mainly in the genetics of human populations, producing with Walter Bodmer a comprehensive survey of the subject in their Genetics, Evolution and Man (1976).

He also did much to show how genetic data from present human racial groups could be used to reconstruct their past separations. This reconstruction, based on the analysis of 58 genes, yields a bifurcated evolutionary tree with Caucasian and African races in one branch and Orientals, Oceanians, and Amerinds in the other. The main division appeared, according to Cavalli-Sforza, some 35–40,000 years ago.