Three vespertilionid bats were captured from Amami-Oshima Island, and had an average forearm length of 35 mm and average body weight of 8 g. From the side of the bat skulls, the ridge of the crown was slightly curved at the base of the snout; the second upper premolar (P²) in the bats was relatively large; the male baculum was shaped like an elongated, 3-mm-long spear tip and was very similar in both shape and size to that of Hypsugo pulveratus. The phylogenetic tree inferred from the mitochondrial COI gene sequences revealed that these bats clustered with H. pulveratus. Therefore, the bats from Amami-Oshima Island were identified as H. pulveratus. The present paper lists and depicts the morphological differences between H. pulveratus and H. alaschanicus so far known from the territory of Japan. Finally, genetic analysis based on the barcoding gene sequences revealed that these species formed distinct clades.