Fish assemblage structure was evaluated and compared among 36 karstic pools located within protected areas of the Calakmul Biosphere Reserve (southern Mexico) and unprotected adjacent areas beyond the Reserve. Nonmetric multidimensional scaling (MDS), indicator species analysis (ISA), and canonical correspondence analysis (CCA) were used to identify which environmental factors reflected local influences and to evaluate the correlation of these variables with fish assemblages structure. Thirty-one species were encountered in these karstic pools, some for the first time within the Reserve. These aquatic environments were separated into three groups based on physico-chemical characteristics. Although CCA identified significant associations between several fish species (based on their relative abundance) and environmental variables (K, NH4, NO3, and conductivity), the most abundant species (Astyanax aeneus, Poecilia mexicana, and Gambusia sexradiata ) occur in most pools and under several environmental conditions. Baseline data on fish diversity along with a continued monitoring program are essential in order to evaluate the conservation status of fish assemblages and their habitats, as well as to measure the influence of anthropogenic impacts on pristine habitats such as the karstic pools of the Calakmul Biosphere Reserve.