Although geneticists have extensively debated the mode by which agriculture diffused from the Near East to Europe, they have not directly examined similar agropastoral diffusions in Africa. It is unclear, for example, whether early instances of sheep, cows, pottery, and other traits of the pastoralist package were transmitted to southern Africa by demic or cultural diffusion. Here, we report a newly discovered Y-chromosome-specific polymorphism that defines haplogroup E3b1f-M293. This polymorphism reveals the monophyletic relationship of the majority of haplotypes of a previously paraphyletic clade, E3b1-M35*, that is widespread in Africa and southern Europe. To elucidate the history of the E3b1f haplogroup, we analyzed this haplogroup in 13 populations from southern and eastern Africa. The geographic distribution of the E3b1f haplogroup, in association with the microsatellite diversity estimates for populations, is consistent with an expansion through Tanzania to southern-central Africa. The data suggest this dispersal was independent of the migration of Bantu-speaking peoples along a similar route. Instead, the phylogeography and microsatellite diversity of the E3b1f lineage correlate with the arrival of the pastoralist economy in southern Africa. Our Y-chromosomal evidence supports a demic diffusion model of pastoralism from eastern to southern Africa approximately 2,000 years ago.