Two morphologically distinct forms of the Stonecat Madtom, Noturus flavus, are found in lowland and Eastern Highland drainages of North America. Evolutionary diversity of aquatic organisms in these regions has been attributed to divergence during the Pliocene and Pleistocene epochs followed by dispersal into previously glaciated regions. We surveyed mitochondrial DNA sequence variation across the range of this widely distributed species to determine the evolutionary relationship between the two forms, and to investigate the possible roles of pre-Pleistocene vicariance and post-Pleistocene dispersal on phylogeographic patterns within the species. Analysis revealed four distinct clades, two within the lowland form and two within the highland form, with no geographic overlap. A deep phylogenetic split between the two forms implies historical fragmentation during the Pliocene. Shallow divergences and limited geographic structuring in the lowland form imply recent fragmentation in two glacial refugia during the Pleistocene, followed by dispersal across much of the modern range of the species. The highland form is common to the Cumberland and Tennessee rivers and clades correspond to each drainage, apparently owing to their separation during the Pleistocene. Phylogeographic patterns echo those of other eastern North American stream fishes, with the persistence of deep phylogenetic divergence predicted by the pre-Pleistocene Vicariance Hypothesis and widespread distribution of closely related haplotypes in glaciated regions consistent with the Leading Edge Model of population expansion.