An efficient method was established for detecting the potential aneugenic activity of chemical substances by combining the micronucleus (MN) test with fluorescence in situ hybridization (FISH) technology. Using mouse lymphoma cells (L5178Y tk+/–) and human lymphoblastoid cells (TK6) as models, the genotoxic effects of colchicine, vincristine, and mitomycin C were analyzed. The results showed that more than 70.2% and 50.5% of the micronuclei induced by colchicine and vincristine contained centromeric signals, respectively, indicating that these drugs mainly induced aneuploidy through the mechanism of chromosome loss. In contrast, 85.9% of the micronuclei induced by mitomycin C lacked centromeric signals, confirming that it acted through chromosome breakage. By integrating cellular-level (MN test, formation rate) and molecular-level (FISH, centromere detection) analyses, this method enabled precise discrimination between aneugens and DNA clastogens. This multi-dimensional detection system could significantly improve the accuracy of genotoxicity mechanism determination and hold important application values in fields such as drug safety evaluation, environmental toxicological assessment, and clinical diagnosis.