Guatemala’s Maya Lowlands constitute one of the most biodiverse regions in Mesoamerica and include 35% of the total number of continental fish species estimated for the nation. From May 2000 through February 2001, we conducted the first long-term ichthyological survey of Lachuá Lake, a 4 km2, 195 m deep, karstic sinkhole located in the middle of Guatemala’s Laguna Lachuá National Park (LLNP), southern Maya Lowlands. Thirty-six native fish species were identified and Gobiomorus dormitor Lacepède, 1800 was collected for the first time in northern Guatemala. Greater number of species occurred in the rocky shore of the lake’s littoral zone, especially around the mouths of Lachuá’s tributary and effluent rivers, and close to the visitor center of LLNP. A hierarchical cluster analysis for classifying ichthyogeographically regional fish assemblages placed Lachuá Lake in the upper Usumacinta River drainage basin shared by Guatemala and Mexico, stressing the need for regional conservation and management strategies.