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Background: Designing, implementing, and upscaling effective malaria vector control strategies necessitates understanding of when and where transmission occurs. This study assessed the biting patterns of potentially infectious malaria vectors at various hours, locations, and human behavior in different ecological settings in western Kenya., Methods: Hourly indoor and outdoor catches of human-biting mosquitoes were sampled from 1900 to 0700 hours for four consecutive nights in four houses per village using human landing collection method. The nocturnal biting activities of each Anopheles species were expressed as the mean number of mosquitoes landing per person per hour. The human behavior study was conducted via observations and questionnaire surveys. Species within Anopheles gambiae and Anopheles funestus complexes were differentiated by polymerase chain reaction (PCR) and the presence of Plasmodium falciparum circumsporozoite proteins (CSP) determined by enzyme-linked immunosorbent assay (ELISA)., Results: Altogether, a total of 2,037 adult female Anophelines were collected comprising of A n. funestus s.l . (76.7%), An.gambiae s.l .(22.8%) and Anopheles coustani (0.5%). Overall, Anopheles funestus was the predominant species collected in Ahero (96.7%) while An. gambiae s.l was dominant in Kisian (86.6%) and Kimaeti (100%) collections. PCR results revealed that An. arabiensis constituted 80.5% and 79% of the An.gambiae s.l samples analysed from Ahero and Kisian respectively. An. gambiae s.s (hereafter An.gambiae ) (98.1%) was the dominant species collected in Kimaeti. All the An. funestus s.l samples analysed belonged to An. funestus s.s (hereafter An. funestus ). Indoor biting densities of Anopheles gambiae and An. funestus exceeded the outdoor biting densities in all sites. The peak biting occurred early morning between 0430-0630 hours in the lowlands for An. funestus both indoors and outdoors. In the highlands (Kimaeti), the peak biting of An.gambiae occurred between 0100-0200 hours indoors. Over 50% of the study population stayed outdoors from 1800 to 2200 hours and woke up at 0500 hours coinciding with the times highest numbers of vectors were collected. The sporozoite rate was higher in vectors collected outdoors, with An. funestus being the main malaria vector in the lowlands and An. gambiae in the highland., Conclusion: The study shows heterogeneity of Anophelines distribution, high outdoor malaria transmission, and peak biting activity by An. funestus (early morning) when humans are not protected by bed nets in the lowland sites. Additional vector control efforts targeting the behaviors of these vectors i.e using non-pyrethroids-based indoor residual spraying and spatial repellents outdoors are needed., Competing Interests: Competing interest The authors have declared that no competing interest.