The Río Lerma basin, one of the most important hydrographic regions of Mexico, has a distinctive fish fauna. In recent years, the basin has experienced major increases in human populations and industrial and agricultural development. To assess the health of aquatic ecosystems in the Lerma basin, we analyzed the current status and long-term trends in fish species occurrence at 116 widely distributed sites in relation to water quality and land use information. Our results reveal a staggering and unprecedented level of environmental degradation. Over 50% of our sites are no longer capable of supporting fish life. Many sites have completely disappeared because of groundwater extraction, water diversions, or urbanization. The Alto Lerma subprovince has experienced the greatest negative impacts, but no region of the basin has escaped significant damage. Only 15% of our sites currently support species that we classify as sensitive to environmental degradation, and most of these sites are on small headwater streams or isolated highland lakes that are protected as parks or municipal water supplies. Only one lowland river, the Río Turbio, retains relatively good environmental quality. Forty percent of the 42 species native to the basin have experienced major declines in frequency of occurrence, and three endemic species, Algansea barbata, Chirostoma charari, and Chirostoma compressum may be extinct. Conservation efforts should focus on protecting all sites that still hold native fishes, with particular emphasis on the Río Turbio.