Analysis of mtDNA sequence variation (2,548 bp from ND2, cytb, and part of the control region) indicates that the genus Cyprinodon began diverging in the Late Miocene from a common ancestor with Megupsilon, a monotypic genus on the Mesa del Norte of Mexico. The geographic pattern of mtDNA variation, with estimates of divergence time, suggests that by the end of the Miocene Cyprinodon occurred from the Atlantic Coast and West Indies to near the western margin of North America via ancestral Rio Grande and Colorado River systems. Phylogeographic structure within the major mtDNA complexes supports a variety of hypotheses from geology and previous phylogenetic analyses for Late Neogene connections among basins in southwestern North America now separated by formidable barriers to dispersal. Comparison of the mtDNA tree with previous phylogenetic inferences from allozymes indicates that reticulate evolution involving divergent lineages probably was important in the history of Cyprinodon.