The increasing degradation of freshwater ecosystems has recently demanded the development of methods that allow us to know the significance of the ecosystem alterations. The Indexes of Biological Integrity (IBI) are used as a methodological approach that combines structural and functional elements of aquatic ecosystems. The quantification of biological integrity is obtained by adding the values of measurable ecological attributes of a biological community; related to the structure, composition and function of the ecosystem. However, it is necessary to validate the IBI before its widespread use. The validation consists in the analysis of the relationships among IBI scores and the habitat condition variables. The validation is supported under the premise that water physiochemical conditions and the physical condition of the habitat are the primary factors associated with the assemblages of biological communities of rivers. We validated the Index of Biological Integrity based on macroinvertebrate assemblages (IIBAMA) in 33 sites located in the Lerma and Pánuco River basins. This research represents the first efforts to validate an IBI based on aquatic invertebrate communities within a relatively large area (12 major rivers and five Mexican states) in the country, with the purpose of expanding the area where the IIBAMA could be used to assess the biotic integrity on accurate way.The increasing degradation of freshwater ecosystems has demanded the development of methods that allow us to evaluate the ecosystem alterations. The Indexes of Biological Integrity (IBI) are a methodological approach to assess the condition of aquatic ecosystems. The objective of this study was to estimate the biological integrity and to validate the Index of Biological Integrity based on macroinvertebrate assemblages (IIBAMA) in 33 study sites rivers in the Lerma-Chapala and Pánuco river basins, in central Mexico. The Family-Level Biotic Index (FBI) was used to estimate the water quality and the Visual-Based Habitat Assessment (VBHA) was used to estimate the habitat quality. Spearman correlation analysis among IIBAMA, FBI, VBHA and water characteristics were made for validation of the IIBAMA. Besides, all variables were evaluated together by multivariate analyses. The rivers were classified in three of four biotic integrity categories, poor, regular and good, being poor the most common (88% of the study sites). We did not find study sites with excellent biotic integrity. The rivers of the Lerma-Chapala River Basin showed a worse ecological condition compare with the Pánuco Rivers Basin. We validated and recommended the using of the IIBAMA to assess the biological integrity of these two basins and rivers in central Mexico. This research represents the first efforts to validate an IBI based on aquatic invertebrate communities in a broad scale in Mexico and provide a general framework for their widespread use and to approach the validation and implementation of IBIs in other regions with similar conditions.