The Mayan Biosphere Reserve is the largest protected area in Guatemala. Lake Yaxhá is located inside the core zone. Using electrofishing, seines and gillnets we assessed the fish richness and community in 2011. We collected 18 species distributed in seven families, with Cichlidae (seven species) and Poecilidae (five species) the most specious. We evaluated the effectiveness of electrofishing to sample the most important fish in the artisanal fishery in Petén, Petenia splendida, with September being the month with the highest capture per unit of effort. Lake Yaxhá assemblage was similar to the Usumacinta through a hierarchical cluster analysis, despite being located in the Mopan-Hondo basin. Lake Yaxhá is the only system in the area that does not have non-native fish species, emphasizing the importance of conservation of this lentic system in the Mayan Biosphere Reserve.