The present study was carried out in fish <i>Clarias batrachus</i> to evaluate the genotoxicity induced by herbicide pendimethalin and to find out the role of oxidative stress in induced DNA damage. The LC<sub>50</sub> value (96 h) of pendimethalin was determined (3.55 mg/L) and based on this, sub lethal test concentrations were calculated as SL-I (1/20th LC<sub>50</sub>), SLII (1/15th LC<sub>50</sub>) and SL-III (1/10th LC<sub>50</sub>) to which fish were exposed for 30, 45 and 60 days. Maximum DNA damage was observed in fish exposed to highest concentration of herbicide and for a particular concentration maximum damage occurs after 30 days then a time dependent decrease was observed. After 30 days the SOD and CAT activity was found to be significantly increased with respect to the control group while it was significantly decreased after 45 and 60 days as compared to 30 days of exposure. Level of lipid peroxidation in pendimethalin exposed fish followed the same pattern. GR activity remained same for all duration of herbicide exposure. The DNA damage is due to ROS generated by the metabolism of herbicide which is evident by alteration in LPO and anti-oxidant enzyme activities accompanied by incidence of DNA damage in a similar way. It can also be concluded that for pendimethalin genotoxicity testing <i>C. batrachus</i> can be used as model organism and comet assay and micronucleus test as effective tools.