Microchiroptera have diversified into many species whose size and the shapes of the complicated ear and nose have been adapted to their echolocation abilities. Their speciation processes, and intra- and interspecies relationships are still under discussion. Here we report on the geographical variation of Japanese Rhinolophus ferrumequinum and R. cornutus using the complete sequence of the mitochondrial cytochrome b gene to clarify the phylogenetic positions of the 2 species as well as that of Rhinolophidae within the Microchiroptera. We have found that sequence divergence values within each of the 2 species are unexpectedly low (0.07%-0.94%). We have also found that there is no local specificity of their mtCytb alleles. On the other hand, the divergence values for Japanese Microchiroptera (12.7%-16.6%) are much higher than those for other mammalian genera. Similarly, the values among five genera of Vespertilionidae were 20.5%-27.3%. Phylogenetic analysis shows that the 2 species of family Rhinolophidae in the suborder Microchiroptera belong to the Megachiroptera cluster in the constructed maximum parsimony tree. These results suggest that the speciation of Rhinolophidae involved its divergence as an independent lineage from other Microchiroptera, and other microbats might be paraphyletic. In addition, the tree also shows that the order Chiroptera is monophylitic, and the closest group to Chiroptera is the ungulates.