Few explicit hypotheses for the relationships of unionid mussels exist. The absence of explicit phylogenetic hypotheses is problematic and is in part responsible for the lack of taxonomic stability seen in this group. In this paper we examine the relationships of mussels in the genus Potamilus, based upon the DNA sequences of a 600 base pair portion of the first subunit of the mitochondrial cytochrome c oxidase (COI) gene. We also examine the genetic distinctiveness of populations of the inflated heelsplitter P. inflatus. The molecular phylogeny indicates that Potamilus is paraphyletic with Leptodea fragilis and Lampsilis ornata nested between P. capax and the remaining Potamilus species. With the exception of P. capax, the remaining Potamilus species are depicted as monophyletic and form three distinct clades: (1) a reciprocally monophyletic P, inflatus clade; (2) a P. ohiensis/P. amphichaenus clade; and (3) a P. Purpuratus/P. p. coloradoensis/P. alatus clade. While bootstrap values indicate a high degree of support for these three clades, relationships among these three clades are not as strongly supported. The genetic distinctiveness of two populations of the inflated heelsplitter exceeds that seen between some other species in the genus. These populations represent geographically isolated, genetically distinct entities, and we therefore recommend the recognition of both the Amite and the Black Warrior populations of P. inflatus as separate species.