The continued loss of freshwater unionid mussel species in the Great Lakes region, and particularly from Lake Erie, raises the question of how much phylogenetic and genetic variation remain in the watershed. The introduction of molecular markers into population biology offers one reproducible technique for assessing this variation. A fragment of the 16S rRNA gene of mitochondrial DNA has previously been used to infer phylogenetic relationships in the family Unionidae. Therefore, we reanalyzed these published molecular data with the addition of 12 species from the Cuyahoga and Rocky rivers in northeast Ohio. Both rivers drain to Lake Erie. The species assessed were Pyganodon grandis, Lasmigona complanata, Lasmigona compressa, Lampsilis radiata luteola, Fusconaia flava, Potamilus alatus, Strophitus undulatus, and Toxolasma parvus, for which published data were unavailable, and northern specimens of four wide ranging species, Lasmigona costata, Leptodea fragilis, Utterbackia imbecillis, and Quadrula quadrula. The resulting phylogenetic tree remained consistent with the accepted major taxonomic divisions in the Unionidae, and it indicates that these rivers still contain a phylogenetically diverse assemblage. However, the most abundant species all are members of the Anodontinae. Intraspecific variation in these unionid species was low, even where results were compared to published sequences on individuals from distant watersheds. One exception was a unique haplotype of Q. quadrula that differed at more base sites than is generally found for many congeneric species.