An assessment of DNA integrity in erythrocytes using the alkaline comet assay was carried out to estimate the impacts of water pollution on Balkan loaches (Cobitis elongata Heckel et Kner, 1858) inhabiting the Sava and Kupa Rivers, rivers of varying different water quality. The amount of DNA damage in cells was estimated from three different parameters comet tail length as the extent of genetic material migration, tail intensity (% DNA in comet tail) and tail moment. The results suggest a genotoxicity of the aquatic environment in the Sava River and demonstrated significantly lower levels of DNA damage in fish captured from the Kupa River. This study confirmed that the comet assay, applied to fish erythrocytes, is a useful tool in determining potential genotoxicity of water pollutants. Although a good DNA damage pattern for Balkan loach was obtained, due to its global and regional conservation status, only restricted use of a small number of specimens per sampling site could be permitted.