The endangered winged mapleleaf, Quadrula fragosa once occurred in the Mississippi, Tennessee, Ohio, and Cumberland River basins in the United States. While the range of this species was thought to have been reduced to a single population in the St. Croix River (Minnesota/Wisconsin), individuals morphologically similar to Q. fragosa were recently recorded in Missouri, Arkansas, and Oklahoma. We employed a phylogenetic analysis of mitochondrial ND1 gene sequences. Our results indicate that Q. fragosa is a separate species and that the southern populations are genetically Q. fragosa. In addition, we developed and utilized 12 microsatellite loci to quantify genetic variation within and population structure across individuals sampled from the five populations. Results indicate a high level of genetic variation within populations, coupled with excess homozygosity, and significant genetic divergence between populations. These results are interpreted for conservation strategies under consideration by the United States Fish and Wildlife Service and United States Army Corps of Engineers.