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Deciphering the evolutionary forces controlling insecticide resistance in malaria vectors remains a prerequisite to designing molecular tools to detect and assess resistance impact on control tools. Here, we demonstrate that a 4.3kb transposon-containing structural variation is associated with pyrethroid resistance in central/eastern African populations of the malaria vector Anopheles funestus. In this study, we analysed Pooled template sequencing data and direct sequencing to identify an insertion of 4.3kb containing a putative retro-transposon in the intergenic region of two P450s CYP6P5-CYP6P9b in mosquitoes of the malaria vector Anopheles funestus from Uganda. We then designed a PCR assay to track its spread temporally and regionally and decipher its role in insecticide resistance. The insertion originates in or near Uganda in East Africa, where it is fixed and has spread to high frequencies in the Central African nation of Cameroon but is still at low frequency in West Africa and absent in Southern Africa. A marked and rapid selection was observed with the 4.3kb-SV frequency increasing from 3% in 2014 to 98% in 2021 in Cameroon. A strong association was established between this SV and pyrethroid resistance in field populations and is reducing pyrethroid-only nets' efficacy. Genetic crosses and qRT-PCR revealed that this SV enhances the expression of CYP6P9a/b but not CYP6P5. Within this structural variant (SV), we identified putative binding sites for transcription factors associated with the regulation of detoxification genes. An inverse correlation was observed between the 4.3kb SV and malaria parasite infection, indicating that mosquitoes lacking the 4.3kb SV were more frequently infected compared to those possessing it. Our findings highlight the underexplored role and rapid spread of SVs in the evolution of insecticide resistance and provide additional tools for molecular surveillance of insecticide resistance. Author summary: Anopheles funestus is an important malaria transmitting mosquito whose control relies on pyrethroid insecticides. The ability to control malaria transmitting mosquitoes is threatened by the emergence of insecticide resistance. Developing effective strategies to prevent, slow, or overcome resistance require an understanding of the underlying genetic mechanisms. In this study, we identified a transposon containing insertion in Anopheles funestus mosquitoes in Central/East Africa but absent in other regions. Tracking its distribution and frequency over the years revealed a strong selection with frequencies reaching fixation in less than 3 years. Phenotype/genotype association studies revealed a strong correlation between pyrethroid resistance and genetic variant genotype but negative correlation with plasmodium parasite infection in mosquitoes. These findings shed light on the often-overlooked role of structural variations in the evolution of insecticide resistance, offering valuable insights and tools for monitoring and addressing this critical issue in malaria control. [ABSTRACT FROM AUTHOR]