One of the major problems affecting the ecosystem health is the exposure of heavy metals due to anthropogenic activities. These exposures may induce toxiogenetic damage and carcinogenesis in aquatic organism. DNA damage biomarkers for fish species detect genotoxic parameters for ecological risk assessment. In the present study, the effect of heavy metals (Cr, Cd, Cu, Fe and Mn) on DNA damage and oxidative stress of Clarias gariepinus was examined by Comet assay at Reference site and two different sampling sites of the Orontes River. Moreover, the relationship between DNA damage and physiological response to oxidative stress caused in C. gariepinus was investigated by sampling seasonal water and fish samples for one year. In this study, Cr, Cd and Cu in water column of Orontes River and the concentrations of Fe and Mn in liver tissue of C. gariepinus were highly exceed the maximum permissible limit set by EPA (1999, 2016) WHO (1989) and TEG (2012) that can cause adverse effects on environment health. SOD activities in liver tissues were significantly higher than those in muscle tissues. SOD, CAT activity and MDA levels of both Site 1 and Site 2 at Orontes River were higher than those of the Reference site. In result of the COMET analysis, the highest levels of DNA damage were found at gill and liver tissues in Site 2 (17.746 ± 1.072% T-DNA; 16.014 ± 0.710% T-DNA respectively) at Orontes river. A higher level of DNA damage in gill cells compared to liver cells was observed in all regions of the Orontes River. In Principal component analysis (PCA), the heavy metals Cd, Cr and Cu (in the water column) and MDA in liver and muscle revealed strong contribution to the observed DNA damage that were scattered opposite to each other's along the principal components. Moreover, correlations between parameters revealed a positive significant relationship between Cd, Cr and Cu in water and DNA damage levels in C. gariepinus. Pearson correlation analysis detected a positive relationship between MDA, CAT and SOD and DNA damage levels in African catfish. Consequently, this study exposed genotoxic damage and oxidative stress of C. gariepinus due to metal pollution in Orontes River and lead to the better understanding of genotoxicty, oxidative stress and heavy metal relationships.