Background: The use of insecticides, through indoor residual spraying and long-lasting insecticide-treated nets (LLINs), is essential to control malaria vectors. However, the sustainability of these tools is challenged by the spread of insecticide resistance in Anopheles mosquitoes. This study was conducted to assess the susceptibility to insecticides and to determine the resistance mechanisms in malaria vectors in Dielmo, a rural area of western Senegal where LLINs were introduced a decade ago. Methods: CDC bottle bioassays were used to determine the susceptibility of 2-5 day-old unfed Anopheles gambiae s.l. females to alphacypermethrin (12.5 mu g/bottle), deltamethrin (12.5 mu g/bottle), etofenprox (12.5 mu g/bottle), lambdacyhalothrin (12.5 mu g/bottle), permethrin (21.5 mu g/bottle), DDT (100 mu g/bottle), bendiocarb (12.5 mu g/bottle), pirimiphos-methyl (20 mu g/bottle) and fenitrothion (50 mu g/bottle). The involvement of glutathione-S-transferases (GSTs) in insecticide resistance was assessed using a synergist, etacrynic acid (EA, 80 mu g/bottle). Polymerase chain reaction (PCR) was used to investigate the presence of 'knock-down resistance (kdr)' mutation and to identify sibling species within the An. gambiae complex. Results: CDC bottle bioassays showed that mosquitoes were fully susceptible to lambdacyhalothrin, bendiocarb and fenitrothion. Overall, mortality rates of 97, 94.6, 93.5, 92.1, and 90.1% were, respectively, observed for permethrin, deltamethrin, pirimiphos-methyl, etofenprox and alphacypermethrin. Resistance to DDT was observed, with a mortality rate of 62%. The use of EA significantly improved the susceptibility of An. gambiae s.l. to DDT by inhibiting GSTs (p = 0.03). PCR revealed that Anopheles arabiensis was the predominant species (91.3%; IC 95 86.6-94%) within An. gambiae complex from Dielmo, followed by Anopheles coluzzii (5.4%; IC 95 2.7-8.1%) and Anopheles gambiae s. s. (3.3%; IC 95 0.6-5.9%). Both 1014F and 1014S alleles were found in An. arabiensis population with frequencies of 0.08 and 0.361, respectively, and 0.233 and 0.133, respectively in An. coluzzii. In An. gambiae s. s. population, only kdr L1014F mutation was detected, with a frequency of 0.167. It was observed that some individual mosquitoes carried both alleles, with 19 specimens recorded for An. arabiensis and 2 for An. coluzzii. The presence of L1014F and L1014S alleles were not associated with resistance to pyrethroids and DDT in An. arabiensis. Conclusions: The co-occurrence of 1014F and 1014S alleles and the probable involvement of GSTs enzymes in insecticide resistance in An. gambiae s.l. should prompt the local vector programme to implement non-pyrethroid/DDT insecticides alternatives.