The phylogenetic relationships of species of the mussel genus Ptychobranchus Simpson, 1900 were examined using the mitochondrial DNA sequences of the ND1 and 16S gene regions. A total of 31 individuals representing the five species are included in this analysis. Outgroups were drawn from other unionid genera previously shown to be closely related to Ptychobranchus. Phylogenetic analysis was conducted using Bayesian methods applying several data-partitioning strategies. The results of all analyses support the monophyly of Ptychobranchus, and the interrelationships of its constituent species are consistent across all analyses. Ptychobranchus occidentalis (Conrad, 1836) is recovered as paraphyletic with P. fasciolaris (Rafinesque, 1820). Molecular analyses indicate that Ptychobranchus jonesi van der Schalie, 1934 is a member of the genus, and character state reconstruction predicts that it should possess the complex conglutinate that is found in other species of Ptychobranchus.