A repeated pattern in North American fishes is species having geographic ranges consisting of two disjunct, widely separated areas of distribution, one in previously glaciated northern areas and one in the unglaciated Ozarks. We used phylogenetic analyses of mitochondrial DNA (ND2 and cyt b) and a nuclear DNA sequence (S7 intron 1) to examine the historical biogeography of one such species, the Least Darter, Etheostoma microperca. Uniquely for Ozark fishes, populations in western Ozark tributaries of the Arkansas River represent most of the evolutionary legacy of the species, with three markedly divergent cyt b clades. Populations in the Illinois River (Arkansas) and Shoal Creek (Missouri) appear to have been isolated for about 5 and 2 my, respectively, and potentially represent undescribed species. A wide-ranging northern clade (Hudson Bay, Great Lakes, and Ohio and upper Mississippi river systems) was sister to a clade comprising haplotypes from Blue River and all Ozark haplotypes exclusive of the Illinois River and Shoal Creek clades. The northern clade appears to have survived Illinoian and Wisconsin glaciations in one or more northern refuges and exhibits molecular genetic signals of population expansion during the Sangamon interglacial. The Blue River and western Ozark populations are especially susceptible to local extirpation with little opportunity for replacement. Persistence of these populations will require maintenance of springflows via regional groundwater management.