Metabolic mechanisms conferring pyrethroid resistance in malaria vectors are jeopardizing the effectiveness of insecticide-based interventions, and identification of their markers is a key requirement for robust resistance management. Here, using a field-lab-field approach, we demonstrated that a single mutation G454A in the P450 CYP9K1 is driving pyrethroid resistance in the major malaria vector Anopheles funestus in East and Central Africa. Drastic reduction in CYP9K1 diversity was observed in Ugandan samples collected in 2014, with the selection of a predominant haplotype (G454A mutation at 90%), which was completely absent in the other African regions. However, 6 years later (2020) the Ugandan 454A-CYP9K1 haplotype was found predominant in Cameroon (84.6%), but absent in Malawi (Southern Africa) and Ghana (West Africa). Comparative in vitro heterologous expression and metabolism assays revealed that the mutant 454A-CYP9K1 (R) allele significantly metabolizes more type II pyrethroid (deltamethrin) compared with the wild G454-CYP9K1 (S) allele. Transgenic Drosophila melanogaster flies expressing 454A-CYP9K1 (R) allele exhibited significantly higher type I and II pyrethroids resistance compared to flies expressing the wild G454-CYP9K1 (S) allele. Furthermore, laboratory testing and field experimental hut trials in Cameroon demonstrated that mosquitoes harboring the resistant 454A-CYP9K1 allele significantly survived pyrethroids exposure (odds ratio = 567, P < 0.0001). This study highlights the rapid spread of pyrethroid-resistant CYP9K1 allele, under directional selection in East and Central Africa, contributing to reduced bed net efficacy. The newly designed DNA-based assay here will add to the toolbox of resistance monitoring and improving its management strategies.