The strong negative anthropogenic pressure on the River Bregalnica resulted in the increase of pollution in the river ecosystem. The aim of this study was to evaluate if aquatic pollutants generate genotoxic effects in barbel (Barbus peloponnesius). Thirty individuals were collected from three localities along the river: one control site and two locations in the middle and lower course of the river with the presence of different pollution sources. Blood smears were stained with May-Grünwald/Giemsa method and the frequency of micronuclei, vacuolated nuclei, binuclei, blebbed nuclei and notched nuclei expressed in per mill was recorded. In fish collected from the control site, the investigated erythrocyte nuclear abnormalities revealed lowest values compared to polluted sites. In general, significantly higher values for erythrocyte deformations, including micronuclei, were noted in the middle and lower part of the river. Significantly higher values of vacuolated and blebbed nuclei were detected in fish from the middle course of the river, while at the lower course the frequencies of occurrence of micronuclei and notched nuclei were significant compared with control site. The middle course of the River Bregalnica has more diverse pollutants compared to the lower course, and consequently the highest frequency of all nuclear deformations was found in barbel from the middle course of the river. Obtained results suggest that pollutants draining into the Bregalnica river ecosystem display a genotoxic effect on the local barbel population.