European inland waters are under continuous threat of anthropogenic pollution. Determination of background level of biomarker response and subsequent classification of the impact increases the applicability of results. In the current study, we evaluate the range of chemical contamination by measuring the concentrations of metals, polycyclic aromatic hydrocarbons and polychlorinated biphenyls, and the levels of environmental genotoxicity by using the micronuclei and nuclear bud tests in bivalve mussels of the major Lithuanian rivers. Second, we aimed to evaluate the association between chemical contamination and genotoxicity biomarker responses. Finally, we set to determine the background level of genotoxic effects. Such value (summed frequency of MN and NB) was assessed—6‰. On that basis, we develop a scale of potential genotoxic impact and perform ranging sites into five categories. The results clearly indicate the existence of significant differences in the levels of chemical pollution and genotoxicity in different sites. Increased levels of studied parameters were assessed at the areas affected by municipal and industrial effluents, road runoff, combustion products, and in the area contaminated by accidental spillage. On the other hand, downstream decrease of contamination level, presumably associated with biological degradation and photochemical oxidation, were also observed. Genotoxicity parameters were associated with PAH and metal concentrations measured in mussel tissues as well as in sediments. Results also indicate that in situ genotoxicity assessment performed in the areas affected by long‐term contamination of municipal origin might be not sufficiently precise. Study highlights the necessity to combine genotoxicity assessment with chemical analysis. Environ. Mol. Mutagen. 61:338–354, 2020. © 2019 Wiley Periodicals, Inc.