BackgroundMalaria vector control efforts have taken malaria related cases down to appreciable number per annum after large scale of intervention tools. Insecticides-based tools remain the major control option for malaria vectors in Kenya and, therefore, the potential of such programs to be compromised by the reported insecticide resistance is of major concern. The objective of this study was to evaluate the status of insecticide resistance in malaria vectors in different agro ecosystems from western Kenya.MethodsThe study was carried out in the lowlands and highlands of western Kenya namely; Ahero, Kisian, Chulaimbo, Emutete, Emakakha, Iguhu, and Kabula. World Health Organization tube bioassays was conducted using standard diagnostic dosages of Lambdacyhalothrin, Deltamethrin, Permethrin, DDT, Bendiocarb, and Malathion tested on Anopheles mosquitoes collected from seven sites; Ahero, Kisian, Chulaimbo, Emutete, Emakakha, Iguhu, and Kabula. Biochemical assays, where the enzymatic activity of three enzymes (monooxygenases, esterases, and glutathione S-transferases) were performed on susceptible and resistant mosquito populations. Wild mosquito populations were identified to species level using polymerase chain reaction (PCR). The species of the wild mosquito populations were identified to species level using PCR. Real-time PCR was performed on the susceptible and resistant mosquitoes after the WHO tube bioassays to determine the presence of knockdown resistance (kdr) allele.ResultsWHO susceptibility tests indicated that Anopheles gambiae showed resistance to Pyrethroids and DDT in all the study sites, to Bendiocarb in Iguhu and Kabula and susceptible to Malathion (100% mortality) in all the study sites. There was an elevation of monooxygenases and esterases enzymatic activities in resistant An. gambiae mosquito populations exposed to Lambdacyhalothrin, Permethrin, Deltamethrin and DDT but no elevation in glutathione S-transferases. A high frequency of L1014S allele was detected in An. gambiae s.s. population, but there was no kdr allele found in Anopheles arabiensis mosquitoes.ConclusionAn. gambiae mosquitoes from western Kenya have developed phenotypic resistance to pyrethroids and DDT. Therefore, there is a need for further research covering different climatic zones with different agroeconomic activities for detailed report on current status of insecticide resistance in malaria vectors.