The genus Myotis is comprised of about 100 species that are unequally distributed between the Northern (81% of the species) and the Southern hemisphere (19% of the species). Only eight species of Myotis occur in the Ethiopian region, but this is the only biogeographic region with representatives of all four classical subgenera, suggesting a diverse assemblage of morphotypes. We used sequences of a mitochondrial DNA gene (cyt b) to investigate the evolution and the phylogenetic position of seven of the eight Ethiopian species, and compared them to a broad sampling of Myotis from the World and of other vespertilionids. Phylogenetic reconstruction was based on 91 complete sequences representing 79 species of bats. The two endemic southern African species of the subgenus Cistugo were not placed within the genus Myotis, but were basal to the vespertilionid radiation, as suggested by earlier work based on karyology. The remaining Ethiopian species formed a strong monophyletic clade within Myotis, further stressing the importance of biogeography as a good predictor of phylogenetic relationships. This Ethiopian clade includes one Western Palaearctic and one Oriental species, both of which probably secondarily colonized these areas from the Ethiopian region. Molecular dating based on Bayesian inferences suggest that these faunal exchanges occurred at the end of the Miocene, while the split of the Ethiopian clade from the other Old World Myotis dates back to the middle Miocene, quite early in the Myotis radiation. Thus, the relative paucity of species in sub-Saharan Africa cannot be attributed to a late entry into this continent. Instead, these molecular results suggest that other evolutionary processes are responsible for the poor species diversity of Myotis found in Africa today.