BACKGROUNDThe massive use of insecticides in public health has exerted selective pressure resulting in the development of resistance inAedes aegyptito different insecticides in Venezuela. Between 2010 and 2020, the only insecticides available for vector control were the organophosphates (Ops) fenitrothion and temephos which were focally applied.OBJECTIVESTo determine the state of insecticide resistance and to identify the possible biochemical and molecular mechanisms involved in three populations ofAe. aegyptifrom Venezuela.METHODSCDC bottle bioassays were conducted onAe. aegypticollected between October 2019 and February 2020 in two hyperendemic localities for dengue in Aragua State and in a malaria endemic area in Bolívar State. Insecticide resistance mechanisms were studied using biochemical assays and polymerase chain reaction (PCR) to detectkdrmutations.FINDINGSBioassays showed contrasting results among populations; Las Brisas was resistant to malathion, permethrin and deltamethrin, Urbanización 19 de Abril was resistant to permethrin and Nacupay to malathion. All populations showed significantly higher activity of mixed function oxidases and glutathione-S-transferases (GSTs) in comparison with the susceptible strain. Thekdrmutations V410L, F1534C, and V1016I were detected in all populations, with F1534C at higher frequencies.MAIN CONCLUSIONInsecticide resistance persists in threeAe. aegyptipopulations from Venezuela even in the relative absence of insecticide application.