The San Pedro–Mezquital River Basin is located in the southern Sierra Madre Occidental, at the Nearctic– Neotropical transition. The river traverses the Sierra through a canyon that reaches over 1000 m in depth. Based on examination of museum specimens, literature records, and our own collections, we documented the occurrence of 120 species (24.6% of the Mexican terrestrial mammals), 24 endemic to Mexico. Richness was comparable with other megadiverse areas of Mexico, and higher than any other Nearctic–Neotropical transition area, moreover species richness is likely to rise as survey continues. Contrary to expectation, distribution of mammals across the basin not only reflected the Nearctic–Neotropical divide, but a third fauna that is a mixture of tropical, temperate and desert species was identifiable at the canyon. Anthropogenic threats including damming of the river, uncontrolled cattle grazing, and pollution from domestic sources, call for effective management strategies to preserve one of the most biodiverse areas of Mexico.