Various oxidative stress biomarkers in gill, kidney and liver tissues in the Indian freshwater fish Wallago attu (Bl. & Schn.) were investigated. Fish were collected from two sites along the river Yamuna, which differ in their extent and type of pollution load. A comparison was made between the biomarker responses and general water chemistry at the two sites. The oxidative stress biomarkers that were analyzed included superoxide dismutase (SOD), catalase (CAT), xanthine oxidase (XOD) and glutathione redox cycle enzymes viz., glutathione peroxidase (GPx), glutathione reductase (GR) and glucose 6-phosphate dehydrogenase (G6PD). Levels of reduced glutathione (GSH) and lipid peroxidation (LPO) were also evaluated. All biomarkers; SOD (P<0.001 in liver, kidney and gill), XOD (P<0.01 in kidney and P<0.001 in liver and gill), GR (P<0.01 in liver, P>0.05 in kidney and P<0.001 in gill), G6PD (P<0.001 in liver, P>0.05 in kidney and P<0.01 in gill), GSH (P<0.001 in liver, kidney and gill) and LPO (P>0.05 in liver, kidney and gill) were found to be substantially higher in the fish collected from Panipat when compared with values in tissues of fish collected from Agra site. GPx and CAT showed a varied response. GPx activity was higher (P<0.001) in gills and kidney of the fish collected at Panipat site. However, liver showed significant low values (P<0.01) when compared with Agra site values. CAT activity was found to be significantly low, in both liver (P<0.01) and kidney (P<0.001) whereas in gills non-significant (P>0.05) low values were observed. Water chemistry data at two sites indicated that Panipat site with higher biochemical oxygen demand, chemical oxygen demand, pH and low dissolved oxygen was comparatively more polluted than Agra site. Industrial activity profile of both the sites also indicates that Panipat has vigorous industrial activity coupled with intensive use of chemicals in agricultural practices in Haryana state. The findings of the present investigation provide a rational use of oxidative stress biomarkers in aquatic ecosystem pollution biomonitoring. This is also the first such attempt reported from India.