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Background: In sub-Saharan Africa there is widespread use of long-lasting insecticidal nets and indoor residual spraying to help control the densities of malaria vectors and decrease the incidence of malaria. This study was carried out to investigate the resting behavior, host preference and infection with Plasmodium falciparum of malaria vectors in Ghana in the context of the increasing insecticide resistance of malaria vectors in sub-Saharan Africa. Methods: Indoor and outdoor resting anopheline mosquitoes were sampled during the dry and rainy seasons in five sites in three ecological zones [Sahel savannah (Kpalsogo, Pagaza, Libga); coastal savannah (Anyakpor); and forest (Konongo)]. Polymerase chain reaction-based molecular diagnostics were used to determine speciation, genotypes for knockdown resistance mutations (L1014S and L1014F) and the G119S ace1 mutation, specific host blood meal origins and sporozoite infection in the field-collected mosquitoes. Results: Anopheles gambiae sensu lato (s.l.) predominated (89.95%, n = 1718), followed by Anopheles rufipes (8.48%, n = 162) and Anopheles funestus s.l. (1.57%, n = 30). Sibling species of the Anopheles gambiae s.l. revealed Anopheles coluzzii accounted for 63% (95% confidence interval = 57.10–68.91) and 27% (95% confidence interval = 21.66–32.55) was Anopheles gambiae s. s.. The mean resting density of An. gambiae s.l. was higher outdoors (79.63%; 1368/1718) than indoors (20.37%; 350/1718) (Wilcoxon rank sum test, Z = − 4.815, P < 0.0001). The kdr west L1014F and the ace1 mutation frequencies were higher in indoor resting An. coluzzii and An. gambiae in the Sahel savannah sites than in the forest and coastal savannah sites. Overall, the blood meal analyses revealed that a larger proportion of the malaria vectors preferred feeding on humans (70.2%) than on animals (29.8%) in all of the sites. Sporozoites were only detected in indoor resting An. coluzzii from the Sahel savannah (5.0%) and forest (2.5%) zones. Conclusions: This study reports high outdoor resting densities of An. gambiae and An. coluzzii with high kdr west mutation frequencies, and the presence of malaria vectors indoors despite the use of long-lasting insecticidal nets and indoor residual spraying. Continuous monitoring of changes in the resting behavior of mosquitoes and the implementation of complementary malaria control interventions that target outdoor resting Anopheles mosquitoes are necessary in Ghana. [ABSTRACT FROM AUTHOR]