Legacies of ancient riverine systems are often manifest in patterns of genetic diversity within aquatic species. The ancient Teays River, a principal drainage of the eastern United States, engaged in several ephemeral connections with neighboring palaeodrainages prior to and during the Pleistocene, when cyclical glacial advance and retreat reconfigured the region's fluvial systems. This study assayed DNA-sequence diversity at one mitochondrial (mtDNA) and three single-copy nuclear DNA (scnDNA) loci from the Tonguetied Minnow (Exoglossum laurae), a species distributed as four disjunct populations, one each within the Upper Great Miami, Upper Allegheny, Upper Genesee, and New rivers. Mitochondrial DNA variation revealed that the New River harbors the highest diversity (h = 0.73) and that the Tonguetied Minnow is composed of two ancient lineages, a Teays River lineage and a Pittsburgh River lineage. Analyses of the scnDNA loci revealed sharing of alleles among populations of E. laurae and between the Tonguetied Minnow and its only congener, the Cutlip Minnow (E. maxillingua), sampled from the Roanoke and Potomac rivers. The probability of interspecific hybridization in the New and Upper Genesee rivers was estimated as 0.16 and 0.34, respectively, but it is likely that some degree of incomplete lineage sorting contributed to these estimates. Probabilities of interspecific hybridization for Cutlip Minnow were 0.62 and 0.65, for the Roanoke and Potomac rivers, respectively, and might reflect ancient hybridization resulting from stream capture events involving these drainages by the Teays River. Management strategies should focus on maintaining the security of the Pittsburgh River lineage in the Upper Great Miami and Upper Allegheny River drainages. Finally, insights into the Tonguetied Minnow's rather convoluted taxonomic history are few, but genetic variation is inconsistent with subspecies status for Tonguetied Minnow in the Upper Great Miami River drainage.