An evaluation of the poorly understood Cenozoic hydrologic history of the American Southwest using combined geological and biological data yields new insights with implications for tectonic evolution. The Mesozoic Cordilleran orogen next to the continental margin of southwestern North America probably formed the continental divide. Mountain building migrated eastward to cause uplift of the Rocky Mountains during the Late Cretaceous to early Tertiary Laramide orogeny. Closed drainage basins that developed between the two mountain belts trapped lake waters containing fish of Atlantic affinity. Oligocene-Miocene tectonic extension fragmented the western mountain belt and created abundant closed basins that gradually filled with sediments and became conduits for dispersal of fishes of both Pacific and Atlantic affinity. Abrupt arrival of the modern Colorado River to the Mojave-Sonora Desert region at ca. 5 Ma provided a new conduit for fish dispersal. Great dissimilarities in modern fish fauna, including differences in their mitochondrial deoxyribonucleic acid (DNA), indicate that late Miocene runoff from the Colorado Plateau did not flow down the Platte or Rio Grande, or through the Lake Bonneville Basin. Fossil fishes from the upper Miocene part of the Bidahochi Formation on the Colorado Plateau have characteristics that reflect a habitat of large, swift-moving waters, and they are closely related to fossil fishes associated with the Snake and Sacramento Rivers. This evidence suggests that influx of fishes from the ancestral Snake River involved a major drainage, not merely small headwater transfers.