Context: Malaria is one of the deadliest diseases in sub-Saharan Africa. Mosquitoes of the genus Anopheles are the main vectors of malaria. Several methods have been implemented to combat these mosquito vectors, including vector control. This relies on the use of long-lasting insecticide-treated mosquito nets (LLINs) and indoor residual spraying (IRS). In order to update data on the emergence of resistance in vectors, this study was conducted to assess the sensitivity of Anopheles gambiae s.l. to pyrethroids and chlorfenapyr in three departments in southern Benin. Methodology: Larval surveys were carried out in the communes of S？-Ava, Ouidah, Cotonou, Adjarra and Adjohoun in December 2022. The larvae obtained were transported to and reared at the insectarium of the Centre de Recherche Entomologique de Cotonou (CREC). Three- to five-day-old adult females of Anopheles gambiae s.l. were subjected to susceptibility tests in WHO tubes with papers impregnated with deltamethrin (0.05%), permethrin (0.75%), and alpha-cypermethrin (0.05%) and also with the combination of the synergist PBO + deltamethrin and PBO + alpha-cypermethrin. Biological tests in CDC bottles were also carried out on these populations using chlorfenapyr (100 μg). PCR was then used to identify the species of the Anopheles gambiae complex, the frequency of the L1014F and Ace-1 kdr genes and finally the level of expression of detoxification enzymes (esterases, oxidases and Gluthation-S transferases) was determined for each commune. Results: Anopheles gambiae s.l. showed strong resistance to the pyrethroids used, with a mortality rate of less than 90%. On the other hand, absolute sensitivity to the synergists PBO + pyrethroids and to chlorfenapyr was recorded in all the areas studied. PCR revealed two species of the Anopheles complex, with Anopheles coluzzii predominating over Anopheles gambiae in the study areas. In all the sites studied, the frequency of the kdr L1014F resistance allele was very high, while the frequency of the ace-1 resistance allele was low. Biochemical tests showed overexpression of oxidase, esterase and GST activity compared with the susceptible Kisumu strain. Conclusion: Anopheles gambiae s.l showed high resistance to the pyrethroids used and absolute sensitivity to PBO + pyrethroid synergists and chlorfenapyr. In addition, metabolic resistance and Kdr L1014F allele frequency
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