Río Champotón in the Yucatán Peninsula is within the Usumacinta Province, the most diverse in fish fauna in Mexico, and is part of the Mesoamerican hotspot, with high endemism and exceptional habitat loss. The spatial and seasonal variability of its fish fauna and their relation to physical habitat characteristics were studied, finding 53 taxa. Canonical correspondence analysis shows salinity, distance from the river mouth, and substratum type differentiate an estuarine and a freshwater region. Fish fauna replacement was observed, with taxa exclusive to each region: 26 estuarine and 27 freshwater species. Six freshwater species enter the estuarine region when the high river flow depletes salinity. Estuary showed greater seasonal differences in specific richness than the freshwater region, which have sites with higher local specific richness. Despite its small drainage, Río Champotón has a highly diverse fish fauna and some areas are affected by human activity and the hurricane season. Its conservation is of critical importance.