The Yazoo Darter, Etheostoma raneyi (Percidae: subgenus Ulocentra), is a narrowly restricted endemic occurring in small tributaries in the Loessial Hills of the upper Yazoo River basin in northern Mississippi. The range of the species is shared between the Little Tallahatchie and adjacent upper Yocona rivers, but populations in the two rivers are separated by unsuitable habitat in the Mississippi Alluvial Plain. The Chickasaw Darter, Etheostoma cervus, and Firebelly Darter, E. pyrrhogaster, show analogous distributions in the Forked Deer and Obion rivers, respectively, of western Tennessee and Kentucky. Phylogenetic analyses of cyt b and control region mtDNA (1497 sites) data from E. raneyi (n = 12), E. cervus (n = 4), and E. pyrrhogaster (n = 5) recovered two clades of E. raneyi with high bootstrap and decay support that are congruent with localities of specimens from the Little Tallahatchie and Yocona drainages, respectively. Divergence between the clades of E. raneyi was 1.3% (SE = 0.3%). Within drainage divergence was 0.3% (SE = 0.1%) for the Little Tallahatchie clade and 0.1% (SE < 0.1%) for the Yocona clade. Etheostoma cervus and E. pyrrhogaster showed interspecific divergence of 1.3% (SE = 0.2%) and intraspecific divergence of 0.7% (SE = 0.2%) and 0.8% (SE = 0.2%), respectively. These results suggest isolation by vicariance as a mode of speciation in fishes restricted to the Upper Coastal Plain. Conservation action may be in order for E. raneyi as populations from the Little Tallahatchie and Yocona rivers should be treated as separate management units with the latter known from only five small streams, some of which are threatened by encroaching development.