Polluted water in the Chapala–Santiago basin (CSB) contains several genotoxic substances that present risks to human health, particularly among residents of communities surrounding Lake Chapala and the Santiago River, where a high prevalence of cancer has been documented. For this reason, it is necessary to study the genotoxic activity of these waters and the genetic damage in inhabitants of the surrounding populations. This study assessed the genotoxicity of water in various communities in the CSB and evaluated DNA damage to lymphocytes in residents of nearby locations. The alkaline comet assay was employed to evaluate water genotoxicity and DNA damage to lymphocytes in residents living near these waters. A standardized questionnaire was distributed to participants of this study to evaluate their exposure to polluted water. Significant genotoxic activity (p < 0.05) was observed in the lymphocytes of individuals exposed to contaminated water (tail length in Puente Grande 27.88 ± 5.4 compared to 3.77 ± 1.64 of negative control), along with notable DNA damage (p ≤ 0.05) to the lymphocytes of residents living in proximity to these waters (tail length in Juanacatlán 12.3 ± 3.4 compared to 1.4 ± 0.74 of negative control). The waters of the CSB possess the capacity to cause DNA damage; meanwhile, genotoxicity increases from Chapala to El Salto due to the additional input of genotoxic contaminants, thereby elevating the cancer risk for the exposed population. The comet test proved to be a useful tool that allowed data to be obtained quickly and reliably.