Resolving geographic patterns of genetic diversity is necessary for understanding the evolution and biogeography of freshwater biodiversity and has important implications for its conservation and recovery. The Kidneyshell (Ptychobranchus fasciolaris), a freshwater mussel (Bivalvia: Unionidae) distributed throughout the Ohio River watershed and southern portions of the Laurentian Great Lakes watershed. Ptychobranchus fasciolaris is consider secure globally, but it is considered a species of conservation concern in portions of its distribution (e.g., Endangered in Canada). Restriction-site associated DNA sequencing (RADseq) was used to develop a genome-wide dataset of single nucleotide polymorphisms (SNPs) to examine patterns genetic diversity and structure across the range of P. fasciolaris and further used to make inferences about its natural history and biogeography and recommendations for conservation and restoration of populations. We identified two major populations corresponding to the Great Lakes and Ohio River watersheds, indicating that each should be considered a separate management unit. Patterns of genetic variation supported post-Pleistocene colonization of the Great Lakes drainage via probable stream capture of the St. Joseph River from the Wabash River (Ohio River drainage) by the Maumee River (Great Lakes drainage). The lower genetic diversity metrics and effective population size estimates in the Great Lakes population compared to the Ohio River watershed population are indicative of a founder effect and even more evidence of post-Pleistocene colonization of the Great Lakes basin. The lack of clear structure within the Great Lakes and most of the Ohio River watershed indicates that there is, as yet, little evidence of the effects of recent fragmentation on stream systems by dams and habitat degradation.