The Black and Nhue-Day River sub-basins near Hanoi, Vietnam, are crucial aquatic ecosystems that are suffering from severe pollution stemming from industrial, domestic, and agricultural sources, which pose risks to environmental and public health. We assessed water genotoxicity at four locations along a gradient of urbanization in Hanoi and its peripheral regions: a fish farm at Hoa Binh reservoir (HB), a peri-urban fish farm in Phu Xuyen district (PX), and urban lakes Truc Bach (TB) and Thien Quang (TQ). Using the comet assay on Nile tilapia erythrocytes, DNA damage (% tail DNA), reflecting fragmented DNA that migrates out of the nucleus during electrophoresis, demonstrated significant differences between sites (p <
0.001). Urban lakes exhibited lower damage (TB: 16 ± 10%, TQ: 33 ± 17%), while the highest damage levels were observed in the hydropower reservoir (HB: 70 ± 15%). Trace elements (i.e., As, Cd, Cr, Ni, and Pb) analyzed in water did not exhibit a significant correlation with DNA damage, suggesting the presence of other unexamined contaminants, such as pesticides, that may explain these findings. These genotoxicity results emphasize the need for further research to identify the specific origins of the observed DNA damage, such as potential contributions from agricultural runoff, untreated wastewater, or other unexamined contaminants. Understanding these sources is essential for developing targeted water management practices to mitigate environmental risks and ensure the safety of aquaculture products, particularly in areas like the HB reservoir, where fish farming supports food security.