Exposure to pesticides may cause adaptation not only in agricultural pests and pathogens, but also in non-target organisms. Previous studies mainly searched for adaptations in non-target organisms in pesticide-polluted sites. However, organisms may propagate heritable pesticide effects, such as increased tolerance, to non-exposed populations through gene flow. We examined the pesticide tolerance—as one of the pre-assumptions of local adaptation—of the freshwater crustacean Gammarus spp. (at genus level reflecting the gammarid community). The pesticide tolerance was quantified in acute toxicity tests using the insecticide imidacloprid. Gammarids were sampled at pesticide-polluted agricultural sites (termed agriculture), least impacted upstream sites (termed refuge) and transitional sites (termed edge) in six small streams of south-west Germany. Furthermore, we examined the population genetic structure of Gammarus fossarum and the energy reserves (here lipid content) of G. fossarum as well as of Gammarus spp. at the three site types (i.e. agriculture, edge and refuge). We found significantly lower imidacloprid tolerance of Gammarus spp. from agricultural sites compared to edge and refuge sites, potentially due to higher environmental stress at agricultural sites, as indicated by a slightly lower lipid content per mg gammarid tissue. We found no differences in pesticide tolerance between edge and refuge populations, indicating no propagation of pesticide effects to edges. The genetic structure among G. fossarum populations showed significant differentiation between streams, but not within a stream across the site types. We suggest that high gene flow within each stream hindered population differentiation and resulted in similar (pre)adaptations to local stress levels between site types, although they exhibited different pesticide pollution. Further studies on target genes (e.g., conferring pesticide tolerance), population structure and fitness of different phenotypes in particular among non-target organisms are required for adjacent pristine ecosystems to detect potential propagations of pesticide effects.