Species in the scorpion genus Centruroides (Scorpiones: Buthidae) are good candidates for the study of ecological niche partitioning because of their habitat plasticity, widespread geographic distribution, and presence of cryptic species. Currently, three species belonging to three subgroups of Centruroides are distributed along the Isthmus of Tehuantepec in southern Mexico, presenting a rare opportunity to study niche partitioning within a single genus. We examined the environmental, substrate, and habitat conditions of Centruroides flavopictus, Centruroides gracilis, and Centruroides rileyi within Estación de Biología Los Tuxtlas, west of the Isthmus of Tehuantepec. Our results demonstrate habitat partitioning occurs along humidity, temperature, and elevational gradients: individuals of C. gracilis were found in habitats of low humidity and high temperature, and were predominantly found on the substrate in human-mediated habitats; individuals of C. flavopictus were found in a larger range of humidity and temperature conditions within secondary and primary forest; and individuals of C. rileyi had the greatest habitat specificity, and were found in conditions of low temperature and high humidity on trees within primary forest. Furthermore, individuals of C. rileyi were found significantly higher in trees than those of C. flavopictus. This study represents the first example of niche partitioning within a genus of scorpion, and the first description of the ecological niche in an arboreal-specialist scorpion.