Species in the genus Epioblasma have specialized life history requirements and represent the most endangered genus of freshwater mussels (Unionidae) in the world. A genetic characterization of extant populations of the oyster mussel E. capsaeformis and tan riffleshell E. florentina walkeri sensu late was conducted to assess taxonomic validity and to resolve conservation issues for recovery planning. These mussel species exhibit pronounced phenotypic variation, but were difficult to characterize phylogenetically using DNA sequences. Monophyletic lineages, congruent with phenotypic variation among species, were obtained only after extensive analysis of combined mitochondrial (1396 bp of 16S, cytochrome-b, and ND1) and nuclear (515 bp of ITS-1) DNA sequences. In contrast, analysis of variation at 10 hypervariable DNA microsatellite loci showed moderately to highly diverged populations based on FST and RST values, which ranged from 0.12 to 0.39 and 0.15 to 0.71, respectively. Quantitative variation between species was observed in fish-host specificity, with transformation success of glochidia of E. capsaeformis significantly greater (P<0.05) on greenside darter Etheostoma blennioides, and that of E. f. walkeri significantly greater (P<0.05) on fantail darter Etheostoma flabellare. Lengths of glochidia differed significantly (P<0.001) among species and populations, with mean sizes ranging from 241 to 272 µm. The texture and colour of the mantle-pad of E. capsaeformis sensu stricto is smooth and bluish-white, whereas that of E. f. walkeri is pustuled and brown, with tan mottling. Based on extensive molecular, morphological and life history data, the population of E. capsaeformis from the Duck River, Tennessee, USA is proposed as a separate species, and the population of E. f. walkeri from Indian Creek, upper Clinch River, Virginia, USA is proposed as a distinct subspecies.