The Waccamaw Darter Etheostoma perlongum is one of three teleost fish species and several molluscs endemic to Lake Waccamaw, a large Carolina Bay in southeastern North Carolina. We assessed species status by sequencing most of the mitochondrial cytochrome b (cyt b) gene in E. perlongum and its putative sister species, E. olmstedi, from the adjoining Waccamaw River and streams in the nearby Pee Dee and Cape Fear Drainages. Phylogeographic analysis shows strongly supported river- and drainage-specific clades with divergence times dating to the Pleistocene. Haplotypes of Etheostoma perlongum are very similar and not phylogenetically distinct from sequences from the Waccamaw River population of E. olmstedi. However, strong haplotype frequency differentiation shows that free genetic exchange does not occur between the river and lake, and along with life history distinctiveness, suggests reproductive isolation despite contact between these populations. Coalescent simulations showed that incomplete lineage sorting between reproductively isolated populations is a plausible explanation for the failure of cyt b sequences to have achieved reciprocal monophyly. Gene tree/species tree discord equal to that seen in our cyt b data was produced in ≥ 5% of coalescent trees, using lake-river divergence times of T 510 thousand years ago (kya), no gene flow and Ne 10 5; simulations with more realistic values of Ne 104 and T 1020 kya required recurrent lake-river migration to generate comparable levels of incomplete sorting of haplotypes. Studies of behavioral reproductive isolation and multilocus studies of nuclear DNA should be used to distinguish these scenarios and to further evaluate the species status of the endemic Waccamaw Darter.