Percina burtoni (Percidae: subgenus Percina), Blotchside Logperch, historically occupied much of the Tennessee and Cumberland River drainages, but is now restricted to highly fragmented populations where they occur at low densities. Widespread impoundment and subsequent river alteration over the past 60 years has eliminated many populations and reduced the potential for dispersal and gene flow between extant populations. We examined genetic variation at two mitochondrial genes, cytochrome b and ND2, from 74 individuals in ten populations to assess genetic population structure within P. burtoni. These data, as well as the results of recent surveys, were used to assess conservation priorities for this imperiled fish. Phylogenetic analysis recovered two well supported and highly differentiated clades within P. burtoni, one containing individuals from the Duck River drainage and a small tributary to the lower Tennessee River, and the other composed of individuals from the middle and upper Tennessee River drainage. The Duck River clade exhibits lower haplotypic and nucleotide diversity than the Tennessee River clade and shows little geographic partitioning. In contrast, most populations of middle and upper Tennessee River P. burtoni possess unique haplotypes and show strong geographic differentiation. Historical isolation of small populations in tributary systems of the upper Tennessee Basin is hypothesized to be responsible for observed patterns of differentiation within this clade. These results have implications for reintroduction programs and population augmentation with captive-reared individuals. Propagation activities should take fine-scale geographic structure into account prior to any reintroduction activities.