The Goodeinae is a speciose group of viviparous freshwater fishes endemic to the Mesa Central of Mexico. The current taxonomy of the group is based on morphology associated with viviparity and several of the groupings are questionable. We sequenced part of the mitochondrial cytochrome c oxidase subunit I (COI) gene (627bp) and control region (approximately 430bp aligned) of representatives of 36 species (all genera) of goodeid fishes in order to establish phylogenetic relationships among the taxa. Findings support the monophyly of the Goodeidae, the sister-group relationship of the Empetrichthyinae and Goodeinae, and the relationship of Profundulus to the Goodeidae. All goodeine genera but Xenotoca were recovered as monophyletic. Many of the higher-level relationships within the group contradict the findings of previous studies based upon morphology. The rate of molecular change in COI (0.9% per Myr), calibrated with the fossil record and geological data, suggests an approximate age for the Goodeidae of 16.5Myr. The majority of divergence within the Goodeinae appears to have occurred during the Miocene, with subsequent cladogenesis in the Pliocene and Pleistocene. Most recent speciation appears allopatric. River piracy, particularly involving the Rio Ameca basin, has played a significant role in the diversification of the Goodeinae.