Drainage systems of the Great Plains and western Gulf Slope underwent substantial changes through diversions and stream captures during the Pleistocene, either as the result of the glacial advances or through independent geologic processes. The distributions of a variety of fishes that range across west-central North America, such as the plains killifish (Fundulus zebrinus), are thought to be the product of this Pleistocene influence. We examined the geographic pattern of genetic variation in F. zebrinus using three allozyme loci (n = 793), mitochondrial DNA restriction fragment length polymorphisms (RFLPs, n = 352), and sequencing of the mitochondrial cytochrome oxidase I (COI, n = 23) in an attempt to understand the roles of dispersal and vicariance. The phylogeographic patterns were concordant between the allozyme and mitochondrial data with the exception of the population in the North Canadian River. The populations fell into three geographic assemblages, which we designated as northern, central, and southern. A large phylogenetic break (average Roger's D = 0.702; average sequence divergence in RFLPs = 4.6%; average sequence divergence in COI = 5.5%) separated the northern/central and southern assemblages. The northern region was likely colonized sometime during the mid-Pleistocene. Fish in the Brazos and Pecos Rivers probably reached these drainages through stream captures of the Red River. The large phylogenetic break between the northern/central and southern clades supports previous attempts to recognize two species of plains killifish: F. zebrinus and F. kansae.