Etheostoma proeliare is distributed in the Gulf Coastal Plain of southeastern North America from the Colorado River of eastern Texas through the Choctawhatchee River of eastern Alabama, north to the Fall Line, and upstream along the Arkansas River valley into eastern Oklahoma. Parsimony and Bayesian analysis of the ND2 gene from 28 populations recovered a monophyletic E. proeliare (PP: 1.0) containing a basal split between a novel clade of populations in the northern apex of the Mississippi embayment, from the Black and St. Francis rivers to the west of, through the Yazoo River to the east of, the main channel of the Mississippi River, and all other populations (PB:99, PP: 1.0). Southern populations are resolved into three clades: Trinity plus Neches Rivers (PB: 100, PP: 1.0), Sabine River east through western tributaries to the Mississippi River plus the Lake Pontchartrain drainage (PP: 1.0), and eastern tributaries to the Mississippi River east through the Escambia River (PP:0.99). The eastern clade is further divided into Pearl plus Big Black rivers (PB:97, PP: 1.0) and Mobile Basin plus Escambia River clades (PP:0.98). These results indicate that although the mainstem of the Mississippi River corresponds to some phylogeographic breaks in a lowland species, it is not an absolute barrier. Future studies of species in the region should further explore the placement of Lake Pontchartrain populations and the existence of divergent populations in the northern Gulf Coastal Plain.