The Lacanja River, a tropical rain forest river in southern Mexico, remains relatively undegraded and serves as a good reference for comparison with other rivers in the region that are experiencing human impacts. We sampled 12 sites from the headwaters to the mouth of the river (120 km) in both the wet and dry seasons to quantify longitudinal and seasonal patterns in fish community diversity and composition. We collected 44 species (41 native) from the river, with Cichlidae the most speciose family. The most widespread and abundant species wereBrycon guatemalensis, Astyanaxsp. (both Characidae), andCichlasoma intermedium(Cichlidae). Based on fish species composition, the river could be divided into an upper, middle, and lower zone; waterfalls impeded fish movements within and between these zones. The upper zone had the lowest species diversity, and the lower zone had the highest. All of the upper zone fishes also occurred in the lower zone, and community composition changed from upstream to downstream solely through species addition and without species replacement. Overall, species diversity was slightly higher in the dry season.