Freshwater mussels in the family Unionidae have suffered severe population declines because of severe anthropogenic disturbances, such as habitat destruction and habitat alteration. Understanding the genetic diversity of healthy unionid populations is crucial to developing informed management plans for imperilled mussels. Here, we characterize the genetic diversity of two common species, Lampsilis cardium Rafinesque, 1820 and Quadrula pustulosa (I. Lea, 1831), using the mitochondrial gene ND1. Populations of Q. pustulosa contained more numerous and more highly divergent haplotypes than populations of L. cardium. This disparity in genetic diversity could be because of several factors, including differences in population size and location and extent of refugia during the Last Glacial Maximum. For both species, AMOVA analysis indicated no genetic structuring based on location within the river, with genetic diversity concentrated within rather than between populations. This finding is consistent with patterns seen for other common mussel species characteristic of large rivers.