Population structure and fecundity of freshwater mussels can be difficult to assess due to the benthic habitat and complex life cycles of these organisms. However, rapid and reliable classification of unionids can be accomplished with polymerase chain reaction if species-specific DNA primers are established. In this report we describe the sequence analysis of the ITS1 and ITS2 regions from five mussel species (Amblema plicata, Fusconia flava, Lampsilis siliquoidea, Ligumia nasuta, and Pyganodon grandis) isolated from a refuge in Lake Erie. Sequence comparison revealed strong similarities between A. plicata and F. flava and between L. siliquoidea and L.nasuta. P. grandis contained regions of additional DNA not present within the other species. Our study confirmed the placement of Ligumia nasuta within the Lampsilinae. The analysis also revealed DNA sequences within the ITS1 and ITS2 regions that are species-specific. Thus, the development of species-specific PCR primers can be utilized for analysis of adult populations and for glochidia on host fish.