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Limited information from existing data sets and the tremendous amount of diversity in number and kind within the chiropteran family Vespertilionidae (about one-third of all bat species) have hampered efforts to provide adequate assessments of long-standing genealogic hypotheses (e.g., monophyly of the family and of the five subfamilies). We generated approximately 2.6 kilobase pairs of mitochondrial DNA (mtDNA) sequence ecompassing three adjacent genes (12S rRNA, tRNAVal, 16S rRNA) for 120 vespertilionids representing 110 species, 37 of 44 genera, and all subfamilies. We assessed monophyly of Vespertilionidae in initial analyses of 171 taxa including representatives of all bat families (except the monotypic Craseonycteridae), and assessed lower-level relationships by analysis of several truncated taxon sets. Phylogenetic analysis of ribosomal gene sequences provides well-supported resolution for vespertilionid relationships across taxonomic levels. Furthermore, the resolution is not heavily burdened by alignment of ambiguous regions of the ribosomal gene sequences, and topologies and levels of support produced by two phylogenetic methods (Bayesian and Parsimony) agreed markedly. Our analyses suggest relationships that support many parts of the traditional classification but which also support several changes. The majority of these changes also receives support from other data sources, particularly bacular and karyotypic data. We make more than 20 taxonomic conclusions or recommendations and construct a working classification for vespertilionoid bats. Highlights include: Miniopterus (subfamily Miniopterinae) is recognized in its own family, Miniopteridae, as it represents an extremely divergent lineage relative to other vespertilionids, and in some analyses is sister to the molossids and natalids; all other vespertilionids examined form a well-supported clade; two of the traditional subfamilies within Vespertilionidae (sensu stricto) are monophyletic, Murininae and Kerivoulinae; Nyctophilinae has no validity and Vespertilioninae is paraphyletic relative to the position of Myotis; Myotis is sister to a clade containing Kerivoulinae and Murininae and is recognized in its own subfamily, Myotinae; Myotis subgenera Leuconoe, Selysius, and Myotis are polyphyletic, and a subgeneric classification reflecting geography is suggested, broadening subgenus Myotis to include the sampled Old World species, and allocating the sampled New World species to another subgenus (Aeorestes Fitzinger, 1870); Vespertilioninae (excluding Myotis) is monophyletic; Pipistrellus-like bats (i.e., the traditional tribe Vespertilionini) are divided into three tribes (Nycticeiini; Pipistrellini; Vespertilionini); and support for three tribes of Pipistrellus-like bats has several implications at the genus level. Overall, this study offers a robust working hypothesis for vespertilionid relationships and provides a good starting point for new investigations into the evolutionary history of Vespertilionidae.