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Background/Objectives: Significant progress in malaria control has been achieved through long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS), raising hopes for malaria elimination. However, emerging insecticide resistance threatens these gains. This study assessed the susceptibility of Anopheles gambiae s.l. populations to public health insecticides, examined the frequencies of kdr , Ace-1 , and N1575Y mutations, and explored their associations with phenotypic resistance in Dielmo and Ndiop, Senegal. Methods : Anopheles larvae collected between September and December 2022 were reared to adulthood. Adult mosquitoes were exposed to discriminating concentrations of various insecticides following WHO guidelines. Knockdown times (KDT50 and KDT95) for pyrethroids were calculated using the Probit model. RT-qPCR detected target-site mutations ( kdr : L1014F and L1014S , Ace-1 , N1575Y ) and assessed correlations with phenotypic resistance. Species-specific PCR identified species within the An. gambiae complex. Results/Conclusions : The populations of Dielmo and Ndiop showed susceptibility to pirimiphos-methyl and bendiocarb, with no Ace-1 mutation detected. Resistance to DDT and pyrethroids was observed. The knockdown times indicated that alphacypermethrin and lambdacyhalothrin were more effective than permethrin and deltamethrin. The L1014F allele was widespread, while L1014S was absent in Ndiop and low in Dielmo. The N1575Y mutation occurred only in populations with L1014F . The L1014S mutation was significantly associated with resistance to lambdacyhalothrin in both villages and to deltamethrin in Ndiop.