Abstract Aim: To assess the micronuclei assay based on age and gender variability from the buccal mucosal cells in patients using various forms of smokeless tobacco. Materials and Methods: A cross sectional study was conducted among sixty eight individuals who had a history of chewing various forms of smokeless tobacco. The study subjects were divided according to their habits into four groups. The groups were arecanut, betel quid, gutkha and control group. Buccal smears were obtained and the exfoliated cells were stained using Feulgen stain. The micronuclei count was done under 40X magnification and compared among the various groups. Results: One way ANOVA analysis was done to compare the difference in ages of the various groups in the present study. The mean age in years were: control group (21 years), gutkha (30 years), betel quid (50 years) and arecanut chewers (43 years), which was found to be significant (p< 0.01). One way ANOVA analysis was also done to compare the micronuclei values in the various groups and it was found that it was the highest in betel quid chewers (P< 0.05). The comparison of the micronuclei value between control and each of the habits was found to be significant (p< 0.001). Similarly, while comparing the micronuclei value between betel quid and the arecanut was found to be significant (p= 0.003). Conclusion: The frequency of Micronuclei assay formation could be used as markers for early identification of the genotoxic effects of chewing tobacco. Policy implications: Awareness of the toxic effects should be carried out with these target groups in mind.