Populations of Etheostoma tallapoosae (Tallapoosa Darter) have previously been shown to be genetically divergent for mitochondrial DNA. In this study, PCR primers were developed to amplify portions of six nuclear genes, and sequences of these genes were assessed in three of the most divergent Tallapoosa Darter populations. This analysis shows that these populations are also highly divergent for nuclear gene sequences and thus any adaptive or potentially adaptive variation is likely to be partitioned among the populations. Sequences of the six nuclear genes have also been determined in the closely related species E. coosae and E. brevirostrum, and the utility of these nuclear gene sequences to the elucidation of darter phylogeny is discussed.