The Natal long-fingered bat (Miniopterus natalensis) and lesser long-fingered bat (M. fraterculus) are morphologically almost indistinguishable and occur sympatrically over much of their southern African range. This raises the possibility that they are sister taxa. We employed a multidisciplinary approach to examine their taxonomic relationship to one another and to other Miniopterus species, whose global phylogeny requires review. We examined echolocation, morphological, and dietary differences between M. natalensis and M. fraterculus, as well as both nuclear and mitochondrial DNA variation between them in the context of a phylogeny incorporating 13 Miniopterus species and subspecies. Despite similarities in their morphology and distribution, M. natalensis and M. fraterculus echolocate at peak frequencies separated by 12 kHz, and both nuclear and mitochondrial DNA markers confirm they are distinct species. Analysis of cytochrome-b (Cytb) sequences further indicates that M. fraterculus and M. natalensis are not sister taxa; M. fraterculus appears to be more closely related to the greater long-fingered bat (M. inflatus). Examination of the global taxonomy of Miniopterus confirms that Schreibers's long-fingered bat (M. schreibersii) forms a paraphyletic species complex. Furthermore, the miniopterine bats are divided into 2 geographically isolated monophyletic groups, one containing African and European species, and the other taxa from Australasia and Asia. Cytb sequence divergence also suggests that M. natalensis is distinct from the European M. schreibersii. These results support the elevation of M. natalensis to full species rank.