The evolution and spread of insecticide resistance mechanisms amongst malaria vectors across the sub-Saharan Africa threaten the effectiveness and sustainability of current insecticide-based vector control interventions. However, a successful insecticide resistance management plan relies strongly on evidence of historical and contemporary mechanisms circulating. This study aims to retrospectively determine the evolution and spread of pyrethroid resistance mechanisms among natural Anopheles gambiae s.l. populations in Senegal. Samples were randomly drawn from an existing mosquito sample, collected in 2013, 2017, and 2018 from 10 sentinel sites monitored by the Senegalese National Malaria Control Programme (NMCP). Molecular species of An. gambiae s.l. and the resistance mutations at the Voltage-gated Sodium Channel 1014 (Vgsc-1014) locus were characterised using PCR-based assays. The genetic diversity of the Vgsc gene was further analyzed by sequencing. The overall species composition revealed the predominance of Anopheles arabiensis (73.08%) followed by An. gambiae s.s. (14.48%), Anopheles coluzzii (10.94%) and Anopheles gambiae-coluzii hybrids (1.48%). Both Vgsc-1014F and Vgsc-1014S mutations were found in all studied populations with a spatial variation of allele frequencies from 3% to 90%; and 7% to 41%, respectively. The two mutations have been detected since 2013 across all the selected health districts, with Vgsc-L1014S frequency increasing over the years while Vgsc-1014F decreasing. At species level, the Vgsc-1014F and Vgsc-1014S alleles were more frequent amongst An. gambiae s.s. (70%) and An. arabiensis (20%). The Vgsc gene was found to be highly diversified with eight different haplotypes shared between Vgsc-1014F and Vgsc-1014S. The observed co-occurrence of Vgsc-1014F and Vgsc-1014S mutations suggest that pyrethroid resistance is becoming a widespread phenomenon amongst malaria vector populations, and the NMCP needs to address this issue to sustain the gain made in controlling malaria.