A priority issue in ecology and biogeography is understanding the patterns in species diversity and the causal factors of their distribution, which allows the generation of information for conservation strategies. The longitudinal distribution of fishes and their relationships with environmental variables were studied in the Guayalejo-Río Tamesí system (northeastern Mexico) from February 2000 to July 2001. A total of 5918 fish were caught in 27 collections along an altitudinal gradient in the main river course, from high mountain (1500 masl) to coastal plain near Tampico. Forty-three native and five exotic species, belonging to 35 genera in 23 families, were identified. Cluster analyses identified four major fish habitats in the river system. A distinctive euryhaline marine fish habitat (1) occurs near the mouth with native and two exotic species. Two other habitats consist essentially of freshwater fish species that are distributed along the longitudinal gradient. One of these habitats (habitat 4) shows greater diversity, as per the Shannon index value, and also includes amphidromous fish, in addition to two exotic freshwater fish; the other (habitat 2) includes freshwater, euryhaline and three exotic species. The changes in the frequency of occurrence and the abundance of Gambusia vittata, Astyanax mexicanus, and Xiphophorus variatus contribute to explaining differences between these habitats. Another habitat (3) is represented by two sampling sites located near the mouth and consist of freshwater and euryhaline fish and three exotic cyprinids with broad salinity tolerance. The low abundance and richness of exotic species suggest little impact on native fish fauna in this river. The fish assemblage of the Guayalejo-Tamesí river system species changes along a longitudinal gradient with the addition, replacement and presence of indicator species. Upstream fish fauna is mostly composed of freshwater species, some of them generalists that inhabit the entire longitudinal gradient, others that are restricted to certain sites, and the remainder of species is an assemblage composed of a mixture of euryhaline freshwater and marine species near the mouth.