The present study was deal with determination of oxidative stress in fishes of Bisalpur reservoir.. Four areas related to Bisalpur reservoir were selected i.e. Bisalpur, Nasirda, Thadoli and Nagdiya. From each area 20 fishes were collected which are Clarias batrachus (10) and Labeo rohita (10). Markers of oxidative stress were determined in the tissues like gills, liver, kidney and heart. In present study we found that higher catalase activity in thadoli area was probably due to some stressful condition in aquatic medium leading to excessive production of free radicals, overall higher oxidative stress was found in Thadoli area fishes. The present investigation was planned to find out the physiological defense against reactive oxygen species during environmental stress or due to presence of pollutants in water. This investigation was attempted at providing biomarkers of physiological defense against ROS at one platform. This will provide normal range for future ecotoxicological studies and will be helpful in making the strategies to protect the fishes from oxidative stress. The findings of the present investigation will provide a rational use of oxidative stress biomarkers in aquatic ecosystem pollution biomonitoring.