Background: Gutkha is a cheap and convenient betel quid substitute, which is popular among all age groups. Various studies reveal its carcinogenic nature that leads to oral submucosus fibrosis and increases the chances of oral cancer. The micronucleus (MN) assay in exfoliated mucosal cells is a useful method for observing genetic damage in humans. Aim: To observe the genotoxic effect of gutkha on human buccal epithelial cells. Materials and Methods: The MN assay was performed to assess the frequency of MN in human buccal epithelial cells. The study comprises 60 individuals of which 30 individuals were gutkha chewers and another 30 were nonusers (control). The MN frequency was scored to estimate the genotoxic damage. Results: In gutkha users, the frequency of MN was highly significant (17.4 ± 0.944) as compared with nonusers (control) groups (4.53 ± 0.331) (P < 0.001). Conclusions: The MN assay in human buccal epithelial cells is a useful and minimally invasive method for monitoring genetic damage in humans. Asignificantly higher frequency of micronucleated cells are found among gutkha users.