Centruroides baergi is a scorpion species distributed in the biogeographical province of the Balsas Basin in Mexico. Health officials have reported acute envenomation in human populations living on the western side of this scorpion’s range, but none in the eastern region. This disparity in toxicity suggested that there may be two distinct species. We used two different approaches, including venom analysis and morphometric specimens from both regions, to test our hypothesis. We performed chromatographic, electrophoretic, and mass spectrometry analysis to identify the known β-toxins involved in the intoxication. The most remarkable finding was the absence of Cb3 β-toxin in the eastern population. Consequently, the LD50 of the eastern population was lower than that of the western population. We analyzed linear and ratio body measurements with parametric and nonparametric statistics to test species limits. These analyses indicated that all putative populations of C. baergi are significantly similar, suggesting that they may represent a single species. Unexpectedly, the population of scorpions in the center of the study area, Suchixtlahuaca, previously identified as C. baergi, showed significant morphological and venom composition differences. We provided empirical evidence of an abrupt change of highly toxic peptides around the 98th meridian that limits populations of Centruroides baergi to the east of Mexico.