Background: The increasing resistance of malaria vectors to pyrethroids poses a major threat to the effectiveness of vector control interventions in sub-Saharan Africa. The introduction of new-generation insecticides, such as clothianidin and chlorfenapyr, offers a promising strategy for improving the control of resistant mosquito populations. This study aimed to assess the susceptibility status of Anopheles gambiae sensu lato populations to pyrethroids, clothianidin, and chlorfenapyr in northern Benin, while determining species composition and the frequency of the kdr L1014F mutation. Methods: Larvae of Anopheles gambiae s.l. were collected from the communes of Gogounou, Banikoara, and Boukombé between August and October 2024 and reared to adulthood under insectary conditions. Larvae were collected from all types of breeding sites encountered within each commune, without a systematic count of the number of sites sampled; larvae from the different breeding sites of a given commune were pooled prior to rearing and bioassays. Susceptibility to pyrethroids (deltamethrin and alpha-cypermethrin) was assessed using the World Health Organization (WHO) tube bioassay protocol, whereas susceptibility to clothianidin (4 μg/bottle) and chlorfenapyr (100 μg/mL) was evaluated using the Centers for Disease Control and Prevention (CDC) bottle bioassay method. Species within the Anopheles gambiae complex were identified by polymerase chain reaction (PCR), and the kdr L1014F mutation was detected using allele-specific PCR. Results: High levels of pyrethroid resistance were observed in all three study sites, with mortality rates below 60% for all pyrethroids tested. In contrast, complete susceptibility to clothianidin was recorded, with 100% mortality 24 hours after exposure in all mosquito populations. Full susceptibility to chlorfenapyr was also observed, with 100% mortality achieved within 48 hours post-exposure. Of the 149 specimens analyzed, 68.45% were identified as Anopheles gambiae sensu stricto, 30.20% as Anopheles coluzzii, and 1.34% as Anopheles arabiensis. The kdr L1014F mutation was detected at high allelic frequencies, ranging from 83% to 95% among A. gambiae and A. coluzzii, and 75% in the small A. arabiensis subsample (n = 2; 95% CI: 19% - 99%) from Gogounou. Conclusion: Anopheles gambiae s.l. populations from northern Benin exhibit high levels of pyrethroid resistance
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