The ability of Anopheles gambiae mosquitoes to transmit Plasmodium parasites is highly variable between individuals. However, the genetic basis of this variability has remained unknown. We combined genome-wide mapping and reciprocal allele-specific RNA interference (rasRNAi) to identify the genomic locus that confers resistance to malaria parasites and demonstrated that polymorphisms in a single gene encoding the antiparasitic thioester-containing protein 1 (TEP1) explain a substantial part of the variability in parasite killing. The link between TEP1 alleles and resistance to malaria may offer new tools for controlling malaria transmission. The successful application of rasRNAi in Anopheles suggests that it could also be applied to other organisms where RNAi is feasible to dissect complex phenotypes to the level of individual quantitative trait alleles.