A paucity of useful characters, morphological convergence, and potential rapid radiation has hindered systematists in elucidating evolutionary relationships within Vespertilioninae. In this study >8,500 base pairs of digenomic DNA for 111 taxa were sequenced and analyzed using maximum-parsimony and Bayesian phylogenetic methods to construct trees and reexamine hypotheses of supergeneric evolutionary relationships in Vespertilioninae. Results of these analyses validate monophyly of Vespertilioninae with the exclusion of Myotis and support recognition of 6 tribes: Antrozoini, Lasiurini, Scotophilini, Vespertilionini, and 2 new unnamed tribal clades, the perimyotine group and the hypsugine group. Tree topologies indicate a Nycticeiini–Eptesicini group, but this clade is not supported. The heuristically pleasing tribe Plecotini also is unresolved in these analyses. These results provided further support and greater resolution for previously proposed hypotheses of Vespertilioninae evolution based on mitochondrial DNA, and although deep branching patterns are not fully resolved, these data increase our understanding of the evolution of this ecologically important and diverse group of bats.