The Redband Darter, Etheostoma luteovinctum, is a benthic headwater fish species found in the Caney Fork and Stones rivers (Cumberland River) and the Duck and Elk rivers (Tennessee River) of central Tennessee. Phylogenetic analyses of 2601 amplified fragment length polymorphisms (AFLPs) were used to assess the relationships among populations and identified two genetically distinct lineages of Redband Darter. One lineage is comprised of populations in the Duck R., Elk R., Middle Fork Stones R., West Fork Stones R., and Hickory Creek of the Caney Fork River, while the other lineage is confined to the remainder of the known localities in the Cumberland R. drainage. As clades were not restricted to drainage or system boundaries, patterns suggest system and drainage transfers. Additionally, morphological variation was examined among populations using standard meristic data and nuptial male color and pigmentation characteristics. Although some morphological traits varied between the two clades, members were not clearly diagnosable using morphology. Taxonomic recognition is not proposed for the two identified clades; however, each clade should be recognized as an evolutionarily significant unit and regarded as such in future conservation efforts. Furthermore, this study demonstrates the utility of AFLPs to assess genetic variation and phylogenetic relationships for species-level taxonomic or phylogeographic studies.