Both mtDNA and the Y chromosome have been used to investigate how modern humans dispersed within and out of Africa. This issue can also be studied using the JC virus (JCV) genotype, a novel marker with which to trace human migrations. Africa is mainly occupied by two genotypes of JCV, designated Af1 and Af2. Af1 is localized to central/western Africa, while Af2 is spread throughout Africa and in neighboring areas of Asia and Europe. It was recently suggested that Af1 represents the ancestral type of JCV, which agrees with the African origin of modern humans. To better understand the origin of modern Africans, we examined the phylogenetic relationships among Af2 isolates worldwide. A neighbor-joining phylogenetic tree was constructed based on the complete JCV DNA sequences of 51 Af2 isolates from Africa and neighboring areas. According to the resultant tree, Af2 isolates diverged into two major clusters, designated Af2-a and -b, with high bootstrap probabilities. Af2-a contained isolates mainly from South Africa, while Af2-b contained those from the other parts of Africa and neighboring regions of Asia and Europe. These findings suggest that Af2-carrying Africans diverged into two groups, one carrying Af2-a and the other carrying Af2-b; and that the former moved to southern Africa, while the latter dispersed throughout Africa and to neighboring regions of Asia and Europe. The present findings are discussed with reference to relevant findings in genetic and linguistic studies. Am J Phys Anthropol, 2006. © 2005 Wiley-Liss, Inc.