The rivers and streams of west‐central Mexico are becoming increasingly degraded and warrant expanded conservation efforts. We have developed an index of biotic integrity based on fish assemblage characteristics to aid in the preservation of the biological integrity and biodiversity of these waters. Our version is an adaptation of previous versions of the index of biotic integrity, which have proven effective in environmental assessment and monitoring in the United States and elsewhere. It consists of 10 fish assemblage attributes, termed metrics, that are related to environmental quality, ecosystem integrity, and biodiversity in west‐central Mexican streams and rivers: number of native species, percentage of benthic species, number of water column species, number of sensitive species, percentage of tolerant species, percentage of exotic species, percentage of omnivores, percentage of native livebearing species, relative abundance, and percentage of diseased or deformed. Data on fish communities from 27 sites on streams and small rivers in the vicinity of the Sierra de Manantlán, southwestern Jalisco, and in the upper Río Duero basin, northern Michoacán, were used to develop expectations and scoring criteria for each metric. The index value for a site was the sum of the scores for the 10 metrics. Index values and associated ratings of biotic integrity for the 27 sites corresponded closely with independent ratings based on habitat and water quality. However, additional tests of the index with independent data and refinement of metrics and scoring criteria are clearly warranted. The index shows promise for identifying key watersheds for the protection of intact native biotic communities and individual endangered and threatened species and for the long‐term monitoring and evaluation of biodiversity and ecosystem integrity in the streams and rivers of west‐central Mexico. Our version could serve as a model for the development of similar indices for other regions and other types of aquatic ecosystems in Mesoamerica.