We constructed DNA-hybridisation matrices comparing 18 genera of Megachiroptera and an outgroup microchiropteran, and eight species of Pteropus and two related genera. Three species each of Megachiroptera and Microchiroptera, two of Primates, and an outgroup armadillo were compared in another matrix; additional representatives of other mammalian orders figured in a further set of experiments. Among the megachiropterans examined, Nyctimene and Paranyctimene comprise the sister-group to other pteropodids. Of the 'macroglossines', only Macroglossus and Syconycteris are associated apart from typical pteropodines, while the four remaining nectar-feeders (Eonycteris, Megaloglossus, Melonycteris, Notopteris) are independently linked with non-nectar-feeding clades. Thus, Megaloglossus is the nearest relative of Lissonycteris, with Epomophorus and Rousettus successive sister-groups to both, while Eonycteris is the sister of all four; Melonycteris and Pteralopex form a trichotomy with the closely related Acerodon and Pteropus, and Notopteris is the sister-taxon to all four. It therefore appears that anatomical specialisations for nectar- and pollen-feeding evolved (or were lost) several times within Pteropodidae. Cynopterus and Dobsonia represent additional clades within the Pteropodinae, with which Thoopterus and Aproteles are respectively paired. Comparisons among species of Pteropus and related genera suggest that Acerodon may be congeneric with Pteropus, but that Pteralopex clearly is not. The ordinal-level matrices support bat monophyly: No order tested is closer to either of the chiropteran suborders than they are to each other, and bats are separated from Primates by at least two nodes. On the basis of previous rate determinations for mammals, we estimate that the African grouping (Epomophorus, Megaloglossus, Lissonycteris) is mid-Miocene in origin, that the two major pteropodid subfamilies (Nyctimeninae and Pteropodinae, including 'Macroglossinae') separated in the Early Miocene, and that the divergence of chiropteran suborders dates from the latest Cretaceous or earliest Palaeocene. Arrangement of genera within Pteropodidae supports the family's Australo-Pacific or south-east Asian origin.