We used phylogenetic analyses of mitochondrial genes, cytochrome-b, and reduced nicotinamide adenine dinucleotide dehydrogenase 2 (ND2) to test the recent proposal that 3 species of large-footed Myotis (adversus, macropus, and moluccarum) occur in Australia. Analyses show that all Australian populations of large-footed Myotis form a monophyletic group to the exclusion of a group containing Indonesian populations of M. adversus. The haplotype divergence between these 2 groups is high (11.8–12.2%) and is comparable with typical species-level divergences in Chiroptera. Within Australia, 2 recently diverged monophyletic groups of haplotypes are found that are not concordant in geographic distribution with species boundaries based on morphology. Analysis of these data suggests that only a single species of large-footed Myotis occurs in Australia, and because this species is taxonomically distinct from M. adversus in Indonesia, it should be known as M. macropus. Our data also show that 2 species of Myotis occur in Papua New Guinea.