In the central western Senegal, malaria transmission has been reduced low due to the combination of several effective control interventions. However, despite this encouraging achievement, residual malaria transmission still occurring in few areas, mainly ensured by An. arabiensis and An. melas. The resurgence or the persistence of the disease may have originated from the increase and the spread of insecticide resistance genes among natural malaria vectors populations. Therefore, assessing the status and mechanisms of insecticides resistance among targeted malaria vectors is of highest importance to better characterize factors underlying the residual transmission where it occurs. Malaria vectors were collected from three selected villages using nocturnal human landing catches (HLC) and pyrethrum spray collections (PSC) methods. An. gambiae s.l. specimens were identified at the species level then genotyped for the presence of kdr-west (L1014F), kdr-east (L1014S) and ace-1R mutations by qPCR. An. arabiensis (69.36%) and An. melas (27.99%) were the most common species of the Gambiae complex in the study area. Among An. arabiensis population, the allelic frequency of the kdr-east (22.66%) was relatively higher than for kdr-west mutation (9.96%). While for An. melas populations, the overall frequencies of both mutations were very low, being respectively 1.12% and 0.40% for the L1014S and L1014F mutations. With a global frequency of 2%, only the heterozygous form of the G119S mutation was found only in An. arabiensis and in all the study sites. The widespread occurrence of the kdr mutation in both An. arabiensis and An. melas natural populations, respectively the main and focal vectors in the central-western Senegal, may have contributed to maintaining malaria transmission in the area. Thus, compromising the effectiveness of pyrethroids-based vector control measures and the National Elimination Goal. Therefore, monitoring and managing properly insecticide resistance became a key programmatic intervention to achieve the elimination goal where feasible, as aimed by Senegal. Noteworthy, this is the first report of the ace-1 mutation in natural populations of An. arabiensis from Senegal, which need to be closely monitored to preserve one of the essential insecticide classes used in IRS to control the pyrethroids-resistant populations.