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<p>Abstract</p> <p>Background</p> <p>Single nucleotide polymorphisms (SNPs) are the most common source of genetic variation in eukaryotic species and have become an important marker for genetic studies. The mosquito <it>Anopheles funestus </it>is one of the major malaria vectors in Africa and yet, prior to this study, no SNPs have been described for this species. Here we report a genome-wide set of SNP markers for use in genetic studies on this important human disease vector.</p> <p>Results</p> <p>DNA fragments from 50 genes were amplified and sequenced from 21 specimens of <it>An. funestus</it>. A third of specimens were field collected in Malawi, a third from a colony of Mozambican origin and a third form a colony of Angolan origin. A total of 494 SNPs including 303 within the coding regions of genes and 5 indels were identified. The physical positions of these SNPs in the genome are known. There were on average 7 SNPs per kilobase similar to that observed in <it>An. gambiae </it>and <it>Drosophila melanogaster</it>. Transitions outnumbered transversions, at a ratio of 2:1. The increased frequency of transition substitutions in coding regions is likely due to the structure of the genetic code and selective constraints. Synonymous sites within coding regions showed a higher polymorphism rate than non-coding introns or 3' and 5'flanking DNA with most of the substitutions in coding regions being observed at the 3^rdcodon position. A positive correlation in the level of polymorphism was observed between coding and non-coding regions within a gene. By genotyping a subset of 30 SNPs, we confirmed the validity of the SNPs identified during this study.</p> <p>Conclusion</p> <p>This set of SNP markers represents a useful tool for genetic studies in <it>An. funestus</it>, and will be useful in identifying candidate genes that affect diverse ranges of phenotypes that impact on vector control, such as resistance insecticide, mosquito behavior and vector competence.</p>