The family Vespertilionidae comprises the largest number of species in the order Chiroptera, and its members are distributed over most of the world. We collected sequences of the mitochondrial NDI (NADH dehydrogenase subunit 1) gene from 38 species as well as 20 sequences of exon28 of the vWF (von Willebrand Factor) gene and analyzed phylogenetic relationships in Vespertilionidae using maximum-likelihood analyses and SINE (short interspersed elements) insertions. Our data strongly suggest the following: (1) Murininae and Myotis, each of which is classified into different subfamilies, are monophyletic; (2) the monophyly of Murininae, Myotis, Pipistrellini, and Plecotini is supported by our analyses of the NDI and nuclear vWF as well as data from SINE insertions, while Miniopterinae was not included in this group; and (3) Rhinolophoidea (microchiroptera) is separated from all the other microchiropteran species. Our study represents the most comprehensive phylogenetic model of Vespertilionidae to date.