<p><i>Sitophilus oryzae</i> (L.) is a cosmopolitan pest of stored grains, frequently controlled using organophosphates and pyrethroids. However, the continuous use of these insecticides may lead to resistance development. Thus, this study evaluated the evolution of <i>S. oryzae</i> resistance after three generations (F3) of artificial selection with pirimiphos-methyl and bifenthrin, and the persistence of acquired resistance after three additional generations without exposure (F6) to insecticides. Two experiments were conducted: the Continuous Exposure Bioassay (CEB), with exposure to sublethal doses, and the Generation Selection Bioassay (GSB), with more intense selective pressure. Esterase activity was analyzed by electrophoresis to investigate the involvement of these enzymes in resistance mechanisms. Samples were subjected to native PAGE, and esterase bands were visualized using α- and β-naphthyl acetate as substrates. The results showed a significant increase in resistance in both approaches, with evidence of cross-resistance between the insecticides. Isoenzymatic analysis revealed the involvement of esterases, particularly cholinesterase Est-1, whose relative activity was higher in the selected generations, suggesting its role in compound detoxification. Additionally, there was a slight reduction in the resistance ratio after three additional generations without exposure, although not statistically significant. These findings highlight the importance of monitoring enzymatic resistance mechanisms for more effective insecticide management and mitigating the impacts of resistance on pest control strategies.</p>