Recently, populations of Etheostoma tippecanoe from the Tennessee River drainage were described as a new species, Etheostoma denoncourti. Although populations of E. tippecanoe from the lower Tennessee River (Duck and Buffalo Rivers) were assigned to the new taxon, the species description did not contain an analysis of specimens from these rivers. To evaluate the phylogenetic placement of these unanalyzed populations with respect to all other populations of E. tippecanoe and E. denoncourti, we assembled a dataset composed of DNA sequences (complete cytochrome b) from 32 individuals representing all known populations of these species. Parsimony analysis of these populations, 11 other darter taxa, and five outgroup taxa resulted in 3868 equally most-parsimonous trees at 1687 steps. Populations from the Duck and Buffalo Rivers were always resolved as members of the newly described taxon, E. denoncourti, consistent with the original description. Further, E. tippecanoe and E. denoncourti were resolved as monophyletic sister taxa in all most-parsimonious trees. The amount of cytochrome b sequence divergence separating these two taxa (5.63%) is within the range observed for cytochrome b variation between other species of darters.