The aim of the present study was to identify genetic alterations occurring in rotograving workers chronicallyexposed to toluene. A total of 60 samples from 30 exposed subjects and 30 age-matched controls, selected basedon the toluene level present in their urine, were recruited. Exposed subjects were categorized based on theirduration of exposure and smoking habitats. Controls were normal and healthy and categorized based on theirsmoking habits. Cell cultures were established from blood samples collected from the control and experimentalsubjects after obtaining informed consent. G-banding and comet assays were used to identify genetic alterations.A higher degree of total chromosome aberration was identified in exposed subjects compared to controls. Asexpected, controls exhibited minimal number of alterations. The overall CA frequency due to toluene exposurewas significantly different from that of the controls for both chromatid and chromosome type aberrations (P\0.05by ANOVA). . The habit of cigarette smoking among the workers had a synergistic effect on inducing DNAdamage. In conclusion, this work shows a clear genotoxic effect associated with toluene exposure, our resultsalso reinforcing the conclusion of higher sensitivity of cytogenetic assays for the biomonitoring of occupationallyexposed populations. There is a strong need to educate those who work with potentially hazardous materialsabout adverse effects and highlight the importance of using protective measures.