BACKGROUND: Selection pressure from continued exposure to insecticides drives development of insecticide resistance and changes in resting behaviour of malaria vectors. There is need to understand how resistance drives changes in resting behaviour within vector species. The association between insecticide resistance and resting behaviour of Anopheles gambiae sensu lato (s.l.) in Northern Ghana was examined. METHODS: F1 progenies from adult mosquitoes collected indoors and outdoors were exposed to DDT, deltamethrin, malathion and bendiocarb using WHO insecticide susceptibility tests. Insecticide resistance markers including voltage-gated sodium channel (Vgsc)-1014F, Vgsc-1014S, Vgsc-1575Y, glutathione-S-transferase epsilon 2 (GSTe2)-114T and acetylcholinesterase (Ace1)-119S, as well as blood meal sources were investigated using PCR methods. Activities of metabolic enzymes, acetylcholine esterase (AChE), non-specific β-esterases, glutathione-S-transferase (GST) and monooxygenases were measured from unexposed F1 progenies using microplate assays. RESULTS: Susceptibility of Anopheles coluzzii to deltamethrin 24 h post-exposure was significantly higher in indoor (mortality = 5%) than outdoor (mortality = 2.5%) populations (P = 0.02). Mosquitoes were fully susceptible to malathion (mortality: indoor = 98%, outdoor = 100%). Susceptibility to DDT was significantly higher in outdoor (mortality = 9%) than indoor (mortality = 0%) mosquitoes (P = 0.006). Mosquitoes were also found with suspected resistance to bendiocarb but mortality was not statistically different (mortality: indoor = 90%, outdoor = 95%. P = 0.30). Frequencies of all resistance alleles were higher in F1 outdoor (0.11-0.85) than indoor (0.04-0.65) mosquito populations, while Vgsc-1014F in F0 An. gambiae sensu stricto (s.s) was significantly associated with outdoor-resting behaviour (P = 0.01). Activities of non-specific β-esterase enzymes were significantly higher in outdoor than indoor mosquitoes (Mean enzyme activity: Outdoor = : 1.70/mg protein; Indoor = 1.35/mg protein. P < 0.0001). AChE activity was also more elevated in outdoor (0.62/mg protein) than indoor (0.57/mg protein) mosquitoes but this was not significant (P = 0.08). Human blood index (HBI) was predominantly detected in indoor (18%) than outdoor mosquito populations (3%). CONCLUSIONS: The overall results did not establish that there was a significant preference of resistant malaria vectors to solely rest indoors or outdoors, but varied depending on the resistant alleles present. Phenotypic resistance was higher in indoor than outdoor-resting mosquitoes, but genotypic and metabolic resistance levels were higher in outdoor than the indoor populations. Continued monitoring of changes in resting behaviour within An. gambiae s.l. populations is recommended.