Highlights• Seven distinct and geographically coherent bioregions were detected in Northern Middle America based on obligate freshwater fishes.• Biotic regionalization of Northern Middle America is greatly influenced by the presence of three biogeographic barriers: (1) the Sea of Cortés and Sierra de Juárez-Cerro Gordo highlands;(2) the Trans-Mexican Volcanic Belt, Sierra Madre del Sur, and Sierra Madre de Chiapas highlands; and (3) the Río Grande Rift, Sierra Madre Occidental, and Mesa Central highlands.• Species richness across bioregions in Middle America vary from two species in Baja California to 187 in the Grande-Pánuco. Dissimilarity among bioregions ranged from 0.92 to 0.98%, excluding the Baja California bioregion which was 100% different from all other bioregions. Such levels of faunal distinctiveness suggest the presence of strong, longstanding geographical barriers enhanced by aridity in the North.• Our results suggest that the northern boundary of Middle America is not delimited by the political border between the USA and Mexico. Instead, the biological northern border of Middle America extends into the USA drainages in California, the Colorado River basin and the headwaters of the Río Grande in the Southern Rocky Mountains.