Abstract Background Vector control is considered to be the most successful component of malaria prevention programs and a major contributor to the reduction of malaria incidence over the last two decades. However, the success of this strategy is threatened by the development of resistance to insecticides and behavioural adaptations of vectors. The aim of this study was to monitor malaria transmission and the distribution of insecticide resistance genes in Anopheles populations from three rural areas of the Moyen Ogooué Province of Gabon. Methods Anopheles spp. were collected using human landing catches in Bindo, Nombakélé and Zilé, three villages located in the surroundings of Lambaréné, during both the rainy and dry seasons. Mosquitoes were identified morphologically, and DNA was extracted from heads and thoraces. Members of the Anopheles gambiae complex were identified by molecular methods using the PCR SINE200 protocol and by sequencing of the internal transcribed spacer 2 region. Taqman assays were used to determine Plasmodium infection and the presence of resistance alleles. Results Anopheles gambiae sensu lato (97.7%), An. moucheti (1.7%) and An. coustani (0.6%) were the three groups of species collected. Anopheles gambiae sensu stricto (98.5%) and An. coluzzii (1.5%) were the only species of the An. gambiae complex present in the collection. Of the 1235 Anopheles collected, 1193 were collected during the rainy season; these exhibited an exophagic behaviour, and consistently more mosquitoes were collected outdoor than indoor in the three study areas. Of the 1166 Anopheles screened, 26 (2.2%) were infected with Plasmodium species, specifically Plasmodium falciparum (66.7%), P. malariae (15.4%), P. ovale curtisi (11.5%) and P. ovale wallikeri (3.8%). Malaria transmission intensity was high in Zilé, with an average annual entomological inoculation rate (aEIR) of 243 infective bites per year, while aEIRs in Bindo and Nombakélé were 80.2 and 17 infective bites per year, respectively. Both the L1014F and L1014S mutations were present at frequencies > 95% but no Ace1G119S mutation was found. Conclusion Our results demonstrate that malaria transmission intensity is heterogeneous in these three rural areas of Moyen Ogooué Province, with areas of high transmission, such as Zilé. The exophagic behaviour of the mosquitoes as well as the high frequency of resistance mutations are serious challenges that need to be addressed by the deployment of control measures adapted to the local setting. Graphical Abstract