Crystallaria asprella, sole member of the genus Crystallaria, was distributed throughout much of the eastern United States but today persists only in isolated populations. We quantified genetic variation among remaining populations of C. asprella by sequencing the mitochondrially encoded cytochrome b (cyt b) gene in 24 individuals from five sample localities. Phylogenetic analysis of these samples and individuals from eight other darter species, Perca, Gymnocephalus, Stizostedion, and Zingel, resulted in 80 equally most-parsimonious trees. Four of the five populations of C. asprella were monophyletic in all trees. Monophyly of the population from the Cahaba River was supported in 60 of 80 most-parsimonious trees. In the remaining 20 trees, one individual from the Cahaba River was included in a trichotomy with a clade of the other individuals from the Cahaba River and a clade of individuals from the Pearl River. The overall pattern of relationships among populations of C. asprella was (Elk River, WV (Saline River, AR (Zumbro River, MN (Cahaba River, AL, Pearl River, LA)))). The most divergent population of C. asprella is from the Elk River in West Virginia. In spite of considerable collecting effort, only five individuals have been collected from the Elk River since 1987. Crystallaria asprella from the Elk River differ from other populations by 11.2–11.8% sequence divergence. Their continued existence is currently threatened by logging, coal mining, and natural gas exploration. Our data document a distinct evolutionarily significant unit in the Elk River that likely is in great peril and should be protected under the U.S. Endangered Species Act.