Northern Middle America (NoMA) is considered a transition zone between the Nearctic and Neotropical biogeographic realms. In this region, Nearctic and Neotropical freshwater fishes create regional faunas of mixed origin, but their general biogeographic patterns have not been quantified. To identify such patterns, we delineate biogeographic regions (BRs) and major biogeographic barriers of NoMA and summarize patterns of faunal similarity among BRs. We used clustering analysis on a presence-absence matrix of primary and secondary freshwater fishes to group 97 level-6 HydroBASINs units spanning NoMA into BRs. We assessed statistical support of clusters using one-way analysis of similarity and implemented a species-indicator analysis. We delineated biogeographic barriers with the software Barrier 2.2 and determined faunal similarity among BRs using beta diversity-Jaccard dissimilarity and producing a minimum-spanning tree. Seven statistically distinctive and geographically coherent BRs were delineated and described. Barrier analysis identified three major barriers within NoMA. The first barrier combines the Sea of Cortés (Gulf of California) and Sierra de Juárez-Cerro Gordo highlands. The second combines the Trans-Mexican Volcanic Belt, Sierra Madre del Sur, and Sierra Madre de Chiapas highlands. The third combines the Río Grande Rift, Sierra Madre Occidental, and Mesa Central highlands. Faunal dissimilarity was very high among BRs, with lowest dissimilarity (92%) between the Balsas-Nacaome and Grijalva-Usumacinta BRs. Boundaries of NoMA BRs do not correspond with political boundaries. We concluded that bioregions of NoMA are faunally distinct, with limited overlap due to presence of strong, long-standing geographical barriers enhanced by aridity in the North.