We employed restriction site variation in mitochondrial (mt)DNA to determine if significant phylogeographic structure occurs in the North American cyprinid fish Cyprinella lutrensis. Digestion patterns from 16 restriction endonucleases identified fifty mtDNA haplotypes among 127 individuals of Cyprinella lutrensis assayed from localities in the Gulf Coastal Plain, the Great Plains, and the Central Lowlands. Nucleotide sequence divergence among haplotypes was highly variable (mean ± SE: 2.87%± 0.08; range: 0.14–9.24%). Maximum-parsimony analysis and the neighbour joining method of tree construction revealed three major groupings (clades) of haplotypes that differed in geographic distribution. Divergence estimates between the basal clade, comprised of haplotypes primarily from the Brazos River in east Texas, and the remaining two clades, place C. lutrensis in the western Gulf Coastal Plain prior to Pleistocene glaciation. Nucleotide sequence divergence between the second clade, comprised of haplotypes from the Trinity and Calcasieu rivers in east Texas and southwestern Louisiana, respectively, and the third clade (comprised primarily of haplotypes from localities north of Texas and affected directly by Pleistocene glaciation), suggest that C. lutrensis colonized gladated regions to the north during the mid- to late Pleistocene. This hypothesis is supported by levels of intrapopulational nucleotide diversity in geographic localities outside of Texas and by geological evidence. Despite marked geographic variation in morphometries, meristics, and nuptial coloration, mtDNA variation in glaciated regions was not geographically structured, and subspecies of C. lutrensis were not identifiable by phylogenetic analysis of mtDNA.