14Resistance to pyrethroid insecticides is a major concern for malaria vector con-15 trol, because these are the compounds used in almost all insecticide-treated bed-nets 16 (ITNs), and are also widely used for indoor residual spraying (IRS). Pyrethroids target 17 the voltage-gated sodium channel (VGSC), an essential component of the mosquito 18 nervous system, but substitutions in the amino acid sequence can disrupt the activity 19 of these insecticides, inducing a resistance phenotype. Here we use Illumina whole-20 genome sequence data from phase 1 of the Anopheles gambiae 1000 Genomes Project 21 (Ag1000G) to provide a comprehensive account of genetic variation in the Vgsc gene 22 in mosquito populations from eight African countries. In addition to the three known 23 1 resistance alleles, we describe 20 non-synonymous nucleotide substitutions at appre-24 ciable frequency in one or more populations that are previously unknown in Anopheles 25 mosquitoes. Thirteen of these novel alleles were found to occur almost exclusively on 26 haplotypes carrying the known L995F resistance allele (L1014F in Musca domesticus 27 codon numbering), and may enhance or compensate for the L995F resistance pheno-28 type. A novel mutation I1527T, which is adjacent to a predicted pyrethroid binding 29 site, was found in tight linkage with either of two alleles causing a V402L substitution, 30 similar to a combination of substitutions found to cause pyrethroid resistance in several 31 other insect species. We analyse the genetic backgrounds on which non-synonymous 32 alleles are found, to determine which alleles have experienced recent positive selec-33 tion, and to refine our understanding of the spread of resistance between species and 34 geographical locations. We describe twelve distinct haplotype groups with evidence 35 of recent positive selection, five of which carry the known L995F resistance allele, 36 five of which carry the known L995S resistance allele, one of which carries the novel 37 I1527T allele, and one of which carries a novel M490I allele. Seven of these groups 38 are localised to a single geographical location, and five comprise haplotypes from dif-39 ferent countries, in one case separated by over 3000 km, providing new information 40about the geographical distribution and spread of resistance. We also find evidence 41 for multiple introgression events transmitting resistance alleles between An. gambiae 42 and An. coluzzii. We identify markers that could be used to design high-throughput, 43 low-cost genetic assays for improved surveillance of pyrethroid resistance in the field. 44 Our results demonstrate that the molecular basis of target-site pyrethroid resistance 45 in malaria vectors is more complex than previously appreciated, and provide a founda-46 tion for the development of new genetic tools to track the spread insecticide resistance 47 Pyrethroid insecticides have been the cornerstone of malaria prevention in Africa for al-50 most two decades [1]. Pyrethroids are currently used in all insectici...