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The massasauga Sistrurus catenatus was historically divided into three subspecies, but this long-standing taxonomy has recently been called into question. Genetic research now recognizes a split of the species into the eastern massasauga S. catenatus and western massasauga S. tergeminus, with the latter split into two subspecies, the desert massasauga S. t. edwardsii and the prairie massasauga S. t. tergeminus. Although the distinction between geographically isolated populations of S. catenatus and S. tergeminus is well-supported genetically, the geographic relationships among populations of S. t. tergeminus and S. t. edwardsii remain unresolved because of incomplete sampling throughout the species’ range. This poses a difficult challenge for conservation and management of this species. Sistrurus t. tergeminus does not have state or federal conservation status, but S. t. edwardsii has been petitioned for listing under the U. S. Endangered Species Act. In this study, we used nuclear and mitochondrial DNA from 52 individuals from 7 states to explore the taxonomic and geographic relationships between S. t. tergeminus and S. t. edwardsii populations. Maximum likelihood and Bayesian inference frameworks for both nuclear and mtDNA genes indicated that S. t. tergeminus and S. t. edwardsii populations were genetically indistinguishable. However, at the species level, we did find eight wellsupported mtDNA clades within S. tergeminus, including individuals from five peripheral populations in 1) Arizona and western New Mexico, 2) Colorado and Kansas, 3) Missouri, 4) Oklahoma, and 5) southern Texas. These peripherally isolated populations surrounded a larger population of individuals from north-central Texas, New Mexico, and Oklahoma that was contiguous with three additional genetically distinct populations. We conclude that the putative subspecies S. t. tergeminus and S. t. edwardsii, as currently defined, most likely represent polytypic phenotypes of S. tergeminus rather than discrete taxonomic entities. Instead, we suggest that S. tergeminus existed historically as a large, contiguous collection of populations that only recently became fragmented into several, as opposed to two, potentially discrete taxonomic entities.