The genus Myotis includes the largest number of species in the family Vespertilionidae (Chiroptera), and its members are distributed throughout most of the world. To re-evaluate the phylogenetic position of East Asian Myotis species with respect to Myotis species worldwide, we analyzed mitochondrial gene sequences of NADH dehydrogenase subunit 1 and cytochrome b from 24 East Asian individuals as well as 42 vespertilionid bats determined previously. The results suggest that: (1) some individuals having the same species name in Europe and Japan do not form a monophyletic clade, indicating that some bat species exhibit morphological convergence, (2) Japanese Myotis mystacinus forms a sister relationship with Myotis brandtii (Palaearctic), and both species are included in the American clade implying that an ancestor of these species originated in North America, and (3) the Black whiskered bat, Myotis pruinosus, is endemic to Japan and forms sister relationships with Myotis yanbarensis and Myotis montivagus collected from Okinawa (Japan) and Selangor (Malaysia), respectively, implying that M. pruinosus originated from the south. The systematics of Japanese and East Asian Myotis bats were revisited by considering their phylogenetic relationships. Our study provides the first extensive phylogenetic hypothesis of the genus Myotis that includes East Asian and Japanese species.