The Ashy Darter, Etheostoma cinereum (Percidae: subgenus Allohistium), is restricted to flowing pools of relatively silt free, medium-sized rivers in the Cumberland, Duck and Tennessee River drainages in the southeastern United States. Degradation of these habitats within these drainages has fragmented the range of E. cinereum into several disjunct populations and eliminated it from many historical localities. Phylogenetic analysis of cytochrome b sequence from individuals (n = 14) representing each of the extant populations of E. cinereum indicated genetic differentiation among populations inhabiting the Cumberland, Duck, and upper Tennessee River drainages. These analyses are concordant with previously noted patterns of morphological variation and minimally support three different management units (MU) currently recognized as E. cinereum. Efforts to conserve these distinct MUs should be considered in management attempts.