The crystal darter, Crystallaria asprella, exists in geographically isolated populations that may be glacial relicts from its former, wide distribution in the Eastern U.S. An initial phylogeographic survey of C. asprella based upon the mitochondrial cytochrome b (cyt b) gene indicated that there were at least four distinct populations within the species: Ohio River basin, Upper Mississippi River, Gulf coast, and lower Mississippi River. In particular, the most divergent population was the most recently discovered, from the Elk River, WV, in the Ohio River basin, and it was postulated that this population represents an undescribed, potentially threatened species. However, differentiation observed at a single gene region is generally not considered sufficient evidence to establish taxonomic status. In the present study, nucleotide variation at the mitochondrial control region and a nuclear S7 ribosomal gene intron were compared to provide independent verification of phylogeographic results between individuals collected from the same five disjunct populations previously surveyed. Variation between populations at the control region was substantial (except between Gulf drainages) and was concordant with patterns of sequence divergence from cyt b. Only the Elk River population was resolved as monophyletic based upon nuclear S7, but significant differences based upon Φ_ST statistics were observed between most populations. Morphometric data were consistent with molecular data regarding the distinctiveness of the Elk River population. It is proposed that populations of C. asprella consist of at least four distinct population segments, and that the Elk River group likely constitutes a distinct species.