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Background: Changes in the natural habitats of insect groups are determined the genetic polymorphisms between individuals. The objective of this study was to establish the genetic structure of the Anopheles coluzzii populations in four localities of Benin. Methods: Insecticide surveys and larval sampling were conducted on 4 study localities, including Cotonou, Ketou, Zagnanado, and Sô-Ava. Molecular characterizations were performed on the Anopheles mosquitoes collected with the allelic and genotypic frequencies of kdr gene determined. The multiple comparison Chi square test for proportions was performed with R version 3.3.3. Next, the observed heterozygosity, expected heterozygosity, and indices of fixation, and genetic differentiation were estimated. Finally, the Hardy–Weinberg equilibrium (EHW) was determined to assess whether panmixia exists in the different populations of mosquitoes of the agroecological zones under study. Results: Carbamates, pyrethroids, organophosphorus and organochlorines use have been reported in all localities except Sô-Ava. Anopheles coluzzii was strongly represented across all study localities. The L1014F allele was observed in the localities of Kétou, Cotonou and Zagnanado. Likewise, insecticide selection pressure of homozygous resistant individuals (L1014F/L1014F) was significantly higher in Kétou, Cotonou and Zagnanado (p value < 0.05). Surprisingly in Sô-Ava, a relatively high frequency of the L1014F allele despite the reported absence of pesticide use was observed. All mosquito populations were found to be deficient in heterozygosity across the study sites (FIS< 0). No genetic differentiation (FST< 0) was observed in the localities of Zagnanado and Kétou. Conclusion: The survey on the use of insecticides showed that insecticide selection pressures differ across the investigated localities. It would be desirable to rotate or apply formulations of combined products with different modes of action. Doing so would enable a better management of resistant homozygous individuals, and mitigate the resistance effect of commonly used insecticides. [ABSTRACT FROM AUTHOR]