He received a Bachelor of Science degree from the University of Miyazaki in 1953, and published his first article, on the mathematics of plant breeding, that same year.

[1] For the next decade, Nei worked in Japan, including as a research scientist at the National Institute of Radiological Sciences, before emigrating to the United States in 1969.

He was later an Evan Pugh Professor of Biology at Pennsylvania State University and Director of the Institute of Molecular Evolutionary Genetics, working there from 1990 to 2015.

He also showed that, unlike R. A. Fisher's argument, deleterious mutations can accumulate rather quickly on the Y chromosome or duplicate genes in finite populations.

[citation needed] Around 1980, Nei and his students initiated a study of inference of phylogenetic trees based on distance data.

In collaboration with Sudhir Kumar and Koichiro Tamura, he developed a widely used computer program package for phylogenetic analysis called MEGA.

[23] Applying this method, they showed that the exceptionally high degree of sequence polymorphism at MHC loci is caused by overdominant selection.

[25] Nei and his students studied the evolutionary patterns of a large number of multigene families and showed that they generally evolve following the model of a birth–death process.

[16][25] He conducted statistical analyzes of evolution of genes controlling phenotypic characters such as immunity and olfactory reception and obtained evidence supporting this theory.

[25] Masatoshi Nei was born in 1931 Japan, and his lifelong interest in biology and genetics may have its roots in his upbringing on a farm, in a family of farmers.