BACKGROUND: Understanding the interactions between increased insecticide resistance and resting behaviour patterns of malaria mosquitoes is important for planning of adequate vector control. This study was designed to investigate the resting behavior, host preference and rates of Plasmodium falciparum infection in relation to insecticide resistance of malaria vectors in different ecologies of western Kenya. METHODS: Anopheles mosquito collections were carried out during the dry and rainy seasons in Kisian (lowland site) and Bungoma (highland site), both in western Kenya using pyrethrum spray catches (PSC), mechanical aspiration (Prokopack) for indoor collections, clay pots, pit shelter and Prokopack for outdoor collections. WHO tube bioassay was used to determine levels of phenotypic resistance of indoor and outdoor collected mosquitoes to deltamethrin. PCR-based molecular diagnostics were used for mosquito speciation, genotype for knockdown resistance mutations (1014S and 1014F) and to determine specific host blood meal origins. Enzyme-linked Immunosorbent Assay (ELISA) was used to determine mosquito sporozoite infections. RESULTS: Anopheles gambiae s.l. was the most predominant species (75%, n = 2706) followed by An. funestus s.l. (25%, n = 860). An. gambiae s.s hereafter (An. gambiae) accounted for 91% (95% CI: 89-93) and An. arabiensis 8% (95% CI: 6-9) in Bungoma, while in Kisian, An. arabiensis composition was 60% (95% CI: 55-66) and An. gambiae 39% (95% CI: 34-44). The resting densities of An. gambiae s.l and An. funestus were higher indoors than outdoor in both sites (An. gambiae s.l; F1, 655 = 41.928, p < 0.0001, An. funestus; F1, 655 = 36.555, p < 0.0001). The mortality rate for indoor and outdoor resting An. gambiae s.l F1 progeny was 37% (95% CI: 34-39) vs 67% (95% CI: 62-69) respectively in Bungoma. In Kisian, the mortality rate was 67% (95% CI: 61-73) vs 76% (95% CI: 71-80) respectively. The mortality rate for F1 progeny of An. funestus resting indoors in Bungoma was 32% (95% CI: 28-35). The 1014S mutation was only detected in indoor resitng An. arabiensis. Similarly, the 1014F mutation was present only in indoor resting An. gambiae. The sporozoite rates were highest in An. funestus followed by An. gambiae, and An. arabiensis resting indoors at 11% (34/311), 8% (47/618) and 4% (1/27) respectively in Bungoma. Overall, in Bungoma, the sporozoite rate for indoor resting mosquitoes was 9% (82/956) and 4% (8/190) for outdoors. In Kisian, the sporozoite rate was 1% (1/112) for indoor resting An. gambiae. None of the outdoor collected mosquitoes in Kisian tested positive for sporozoite infections (n = 73). CONCLUSION: The study reports high indoor resting densities of An. gambiae and An. funestus, insecticide resistance, and persistence of malaria transmission indoors regardless of the use of long-lasting insecticidal nets (LLINs). These findings underline the difficulties of controlling malaria vectors resting and biting indoors using the current interventions. Supplemental vector control tools and implementation of sustainable insecticide resistance management strategies are needed in western Kenya.