Our extension of the phylogenetic study of Taylor et al. (2009) on a larger, more geographically representative sample confirmed their finding of genetically distinct sympatric lineages of bats currently referred to as Chaerephon pumilus sensu lato (s.l.) (family Molossidae) in southeastern Africa. Chaerephon pumilus s.l. comprised two cytochrome b lineages separated by a mean genetic distance of 0.7% (0.1–1.4%), consistent with intraspecific variability. The C. pumilus s.l. clade was paraphyletic, containing a nested C. leucogaster (Madagascar) clade. As well as the expected four mitochondrial control region lineages, we identified a new strongly-supported clade from the Durban area. Indices of diversity and neutrality, combined with a ragged multimodal mismatch distribution, are inconsistent with demographic expansion of a single C. pumilus s.l. population in southeastern Africa, and suggest that the control region lineages are stable units at demographic equilibrium. Dating analyses suggest that these lineages were established during the late Pleistocene, between 60 000 and 13 000 years ago. We found no evidence to support our hypothesis that the sympatric genetic lineages of C. pumilus s.l. are associated with distinct sonotypes, as discriminant function analysis based on four echolocation parameters could not discriminate between the four clades. We hypothesise that the different genetic lineages may be distinguished by differences in social communication and behaviour.