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Elucidating the complex evolutionary armory that mosquitoes deploy against insecticides is crucial to maintain the effectiveness of insecticide‐based interventions. Here, we deciphered the role of a 6.5‐kb structural variation (SV) in driving cytochrome P450‐mediated pyrethroid resistance in the malaria vector, Anopheles funestus. Whole‐genome pooled sequencing detected an intergenic 6.5‐kb SV between duplicated CYP6P9a/b P450s in pyrethroid‐resistant mosquitoes through a translocation event. Promoter analysis revealed a 17.5‐fold higher activity (p <.0001) for the SV− carrying fragment than the SV− free one. Quantitative real‐time PCR expression profiling of CYP6P9a/b for each SV genotype supported its role as an enhancer because SV+/SV+ homozygote mosquitoes had a significantly greater expression for both genes than heterozygotes SV+/SV− (1.7‐ to 2‐fold) and homozygotes SV−/SV− (4‐to 5‐fold). Designing a PCR assay revealed a strong association between this SV and pyrethroid resistance (SV+/SV+ vs. SV−/SV−; odds ratio [OR] = 2,079.4, p <.001). The 6.5‐kb SV is present at high frequency in southern Africa (80%–100%) but absent in East/Central/West Africa. Experimental hut trials revealed that homozygote SV mosquitoes had a significantly greater chance to survive exposure to pyrethroid‐treated nets (OR 27.7; p <.0001) and to blood feed than susceptible mosquitoes. Furthermore, mosquitoes homozygote‐resistant at the three loci (SV+/CYP6P9a_R/CYP6P9b_R) exhibited a higher resistance level, leading to a far superior ability to survive exposure to nets than those homozygotes susceptible at the three loci, revealing a strong additive effect. This study highlights the important role of structural variations in the development of insecticide resistance in malaria vectors and their detrimental impact on the effectiveness of pyrethroid‐based nets. [ABSTRACT FROM AUTHOR]