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Conservation efforts rely on robust taxonomic assessments that should be based on critical assessment of interspecific boundaries, infraspecific variation, and potentially distinctive peripheral populations. The meadow vole (Microtus pennsylvanicus) is widely distributed across North America, including 28 morphologically defined subspecies and numerous isolated populations. Because some subspecies are of high conservation concern, we examined geographic variation across the range of the species to test existing infraspecific taxonomy in terms of local and regional diversification. We sequenced mitochondrial DNA (mtDNA) from 20 subspecies of M. pennsylvanicus and contextualized infraspecific variation through comparison of pairwise genetic distances derived from an extended data set of 63 species of Microtus. We found strong support for at least three divergent clades within M. pennsylvanicus, with observed intraspecific clade divergence exceeding that between several pairwise comparisons of sister species within Microtus. Six nuclear genes were then sequenced to test the validity of mtDNA structure and to further evaluate the possibility of cryptic, species-level diversity using Bayes factor species delimitation (BFD) analyses. BFD consistently and decisively supported multiple species based on the multilocus approach. We propose that taxonomic revision of the meadow vole is required, with the eastern clade now identified as M. pennsylvanicus (Ord 1815), the western clade as M. drummondii (Audubon and Bachman 1853), and the coastal Florida clade as M. dukecampbelli (Woods, Post, and Kilpatrick 1982). We suggest that such an arrangement would more closely reflect evolutionary history and provide critical context for further examination of distinctive southern peripheral populations that harbor novel evolutionary legacies and adaptive potential.