Insecticide treatments for crops protection in cotton fields have often been cited as the main factor in the selection of insecticide resistance in Anopheles gambiae populations. To explore this hypothesis further, the present study was designed to identify potential practices that may contribute to the emergence of insecticide resistance in Anopheles gambiae in cotton crop protection strategies such as the “Calendar Control Program (CCP)” and “Targeted Intermittent Control Program (TICP)” officially recommended already operate in Benin. Firstly, Knowledge Attitude-Practice (KAP) surveys were organized in the study sites to generate adequate information on the use of insecticide on cotton fields. In each site, leaders of farmer’s organizations were interviewed using semi-structured questionnaires that focused on the treatment strategies, and the use of insecticides in the farms. Further, bioassays were performed on adult mosquitoes collected from various of each program to assess the susceptibility of malaria vectors to insecticide-impregnated papers (permethrin 0.75%, delthamethrin 0.05%, DDT 4%, and bendiocarb 0.1%) following WHOPES guidelines. Results of this study showed that various pesticides particularly pyrethroids were used for pests control in CCP and TICP sites; ii)-Susceptibility tests showed that An. gambiae populations have developped resistance to DDT (4% as mean mortality despite the strategies), permethrin (30% in CCP and TICP), deltamethrin (38% and 46% as mean mortality in CCP and TICP respectivetly), but fully susceptible to bendiocarb . These findings confirmed the role of insecticide treatments in mosquitoes resistance. Measure must be taken for better management of the use of insectides for pest control.