Anthropogenic chemicals in freshwater environments contribute majorly to ecosystem degradation and biodiversity decline. In particular anthropogenic organic micropollutants (AOM), a diverse group of compounds, including pesticides, pharmaceuticals, and industrial chemicals, can significantly impact freshwater organisms. AOM were found to impact genetic diversity of freshwater species; however, to which degree AOM cause changes in population genetic structure and allelic richness of freshwater macroinvertebrates remains poorly understood. Here, the impact of AOM on genetic diversity of the common amphipod Gammarus pulex (Linnaeus, 1758) (clade E) was investigated on a regional scale. The site‐specific AOM levels and their toxic potentials were determined in water and G. pulex tissue sample extracts for 34 sites along six rivers in central Germany impacted by wastewater effluents and agricultural run‐off. Population genetic parameters were determined for G. pulex from the sampling sites by genotyping 16 microsatellite loci. Genetic differentiation among G. pulex from the studied rivers was found to be associated with geographic distance between sites and to differences in site‐specific concentrations of AOM. The genetic diversity parameters of G. pulex were found to be related to the site‐specific AOM levels. Allelic richness was significantly negatively correlated with levels of AOM in G. pulex tissue (p < 0.003) and was reduced by up to 22% at sites with increased levels of AOM, despite a positive relationship of allelic richness and the presence of waste‐water effluent. In addition, the inbreeding coefficient of G. pulex from sites with toxic AOM levels was up to 2.5 times higher than that of G. pulex from more pristine sites. These results indicate that AOM levels commonly found in European rivers significantly contribute to changes in the genetic diversity of an ecologically relevant indicator species.