Previously, braincase shape has been the most useful character for species diagnosis of the Myotis mystacinus group members in Hokkaido, Japan. However, this character is difficult to apply in live individuals. Recently, the different patterns of venation in the tail membrane were described for species diagnosis of M. ikonnikovi and M. mystacinus (actually M. brandtii gracilis in Japan). We evaluated this new method in these two cryptic species in Hokkaido, Japan, based on molecular analysis. Our results revealed complete congruence between haplotypes of the mtDNA cytochrome b gene and the patterns of tail membrane venation in these two species. Thus, tail membrane venation pattern should be regarded as an unambiguous diagnostic character to distinguish M. ikonnikovi and M. b. gracilis under field conditions in Hokkaido. Further, since our field sampling was extensive, we suggested that only two species in the M. mystacinus group, M. ikonnikovi and M. b. gracilis occur in Hokkaido, and concluded that the occurrence of other cryptic species in the group in Hokkaido is unlikely.