The effects that anthropogenic stressors may have on modulating species' plasticity has been relatively unexplored; however, it represents a scientific frontier that may offer insights into their ability to colonize new habitats. To explore the advantage that inhabiting polluted environments may offer to invasive species, we selected the crayfish Procambarus clarkii, a species that can colonize and thrive in a wide range of aquatic environments, including heavily polluted ones. Here, we studied the molecular and physiological responses of crayfish when experimentally exposed to a pesticide mix of azoxystrobin and oxadiazon at sublethal concentrations. We compared these responses in three isolated crayfish populations in Southern France that are established in areas with different pollution levels: i) Camargue, seasonally affected by pesticide pollution; ii) Bages-Sigean, impacted all year-round by domestic effluents and; iii) Salagou, a more pristine site. Gene expression analyses revealed that the response to the pesticide mix was the strongest in the Camargue crayfish. In this population, a total of 88 differentially expressed genes (DEGs) were identified in hepatopancreas and 78 in gills between exposed and control laboratory groups. Among genes that were differentially expressed and successfully annotated, those involved in stress response, DNA repair, immune response, and translation and transcription processes stand out. Interestingly, the hepatopancreas responded mainly with upregulation, while the gills showed downregulation. Our results demonstrate population-specific responses to pesticide stress in populations with different life-history of exposure to pollutants. The high regulation of the aforementioned mechanisms indicates that they play a crucial role in the adaptation of this invasive species to polluted environments.