Bats of the family Pteropodidae, also known as megabats or Old World fruit bats, are widely distributed in tropical areas of Africa, Asia, and Oceania. Of 45 genera in the family, 12 are endemic to the Afro-tropical region and two others have representative species on the African continent. African megabats inhabit wooded habitats and are nearly ubiquitous on the mainland and nearby islands with the exception of desert areas. Some species have been implicated as possible reservoirs of the Ebola Zaire virus. We studied the phylogenetic relationships of mainland African megabats using both mitochondrial and nuclear loci in separate and combined analyses. The phylogenetic trees obtained showed four main African clades: Eidolon, Scotonycterini (including two genera), African Rousettus (three species), and the previously identified ‘endemic African clade’ (nine genera). The latter three lineages form a clade that also includes the Asian species of Rousettus and the Asian genus Eonycteris; Eidolon does not show close relationships to other African genera, instead nesting elsewhere in the megabat tree. Although our results confirm many of the conclusions of previous studies, they challenge the taxonomic status and placement of Epomops dobsonii and Micropteropus, and provide evidence indicating that a new classification at subfamilial and tribal levels is highly desirable. The principal clades we detected represent four independent colonizations of Africa from most probably Asian ancestors. Estimates of divergence dates suggest that these events occurred in different periods and that although local diversification appears to have started in the late Miocene, the more extensive diversification that produced the modern fauna occurred much later, in the Pleistocene.