Three-spined sticklebacks (Gasterosteus aculeatus L.) were collected during different sampling trails from three locations in Northern Germany, which differ in the amount of sewage-treatment effluent that they receive. Due to natural population developments, the size of the specimens caught decreased significantly from April to August. The fish were examined for DNA damage in their blood cells by means of the comet (single-cell gel electrophoresis, SCGE) assay and the micronucleus test (MT). The suitability of stickleback erythrocytes as indicators for genotoxic substances in water was assessed. The median level of strand breakage ranged from 5.23 to 9.67%, and decreased significantly from April to August. The difference between the locations was marginally significant. The amount of micronuclei was more variable (ranging from 0.40 to 4.35%), but appears to better reflect the pollution state of the sampling location. Significant differences between the locations were found. The relatively strong micronucleus induction found in this study may be related to the fish species selected. Contrary to the SCGE results, a significant increase in the number of micronuclei from April to August was observed. A significant negative correlation between strand breakage and micronuclei was found for the sticklebacks from the most polluted location and for the pooled data of all locations. The length of the fish was positively correlated with results of the SCGE and negatively with those of the MT, whereby males show a clearer relation between size and the amount of genotoxic damage. The test results are predominantly affected by seasonal impacts. This study indicates that the outcome of the SCGE and MT applied to sticklebacks is determined by multiple factors, which need to be identified first before these tests can be applied routinely. Because of the profound negative correlation between SCGE and MT results, we recommend to apply both tests for the evaluation of the genotoxic potential of surface waters.