Environmental pollution including mutagens from wastewater effluents and discontinuity by man-made barriers are considered typical anthropogenic pressures on microevolutionary processes that are responsible for the loss of biodiversity in aquatic ecosystems. Here, we tested for the effects of wastewater treatment plants (WWTPs), weirs and other stressors on the invertebrate species Gammarus pulex at the population genetic level combining evolutionary ecotoxicology, body burden analysis and testing for exposure to mutagens. Exposure to chemical pollution alone and in combination with the presence of weirs resulted in a depression of allelic richness in native G. pulex populations. Our results suggest that the input of a mutagenic effluent from a WWTP resulted in a strong increase in private alleles over the affected populations. In addition, the presence of weirs along the river disrupted the migration across the river and thus the gene flow between G. pulex upstream and downstream. This study provides strong evidence that the assessment of genetic variation including private alleles together with the contamination of mutagenic and nonmutagenic chemical pollution offers new insights into the regulation of genetic population structure and highlights the relevance of emerging anthropogenic pressures at the genetic level.