BackgroundHistorically, malaria due toPlasmodium vivaxhas been epidemic in Henan Province, China, withAnopheles sinensisas the main vector. The most effective measures to prevent malaria transmission are based on vector control through the use of insecticides. However, insecticides exert a strong selective pressure on mosquito populations for insecticide resistance. The aim of this study was to investigate the susceptibility profile and population genetic characteristics ofAn. sinensisto provide basic data and scientific guidance for the study of resistance mechanisms and the control ofAn. sinensisin Henan Province.MethodsAdultAnophelesmosquitoes were collected at sites near local farmers' sheepfolds, pigsties and/or cowsheds located in Pingqiao, Xiangfu, Xiangcheng and Tanghe counties/districts of Henan Province during July–September 2021 for insecticide susceptibility testing. Molecular identification of collected mosquitoes as belonging to genusAnopheleswas by PCR, and the frequencies of mutations in the knockdown resistance gene (kdr) and acetylcholinesterase-1 gene (ace-1) were detected using gene amplification. The mitochondrial DNA cytochrome oxidase subunit I (COI) gene was amplified in deltamethrin-resistant and deltamethrin-sensitive mosquitoes to analyze the genetic evolutionary relationship.ResultsA total of 1409Anophelesmosquitoes were identified by molecular identification, of which 1334 (94.68%) wereAn. sinensis, 28 (1.99%) wereAn. yatsushiroensis, 43 (3.05%) wereAn. anthropophagusand four (0.28%) wereAn. belenrae. The 24-h mortality rates ofAn. sinensisin Pingqiao, Tanghe, Xiangcheng and Xiangfu counties/districts exposed to deltamethrin were 85.85%, 25.38%, 29.73% and 7.66%, respectively; to beta-cyfluthrin, 36.24%, 70.91%, 34.33% and 3.28%, respectively; to propoxur, 68.39%, 80.60%, 37.62% and 9.29%, respectively; and to malathion, 97.43%, 97.67%, 99.21% and 64.23%, respectively. One mutation, G119S, was detected in theace-1gene. The frequencies of the main genotypes were 84.21% of specimens collected in Xiangfu (G/S), 90.63% of speciments collected in Xiangcheng (G/G) and 2.44% of speciments collected in Tanghe (S/S). Significantly higher G119S allele frequencies were observed in both propoxur- and malathion-resistant mosquitoes than in their sensitive counterparts in the Tanghe population (P< 0.05). Three mutations, L1014F (41.38%), L1014C (9.15%) and L1014W (0.12%), were detected in thekdrgene. The genotypes with the highest frequency in the populations ofAn. sinensisin Xiangfu and Tanghe were the mutant TTT (F/F) and wild-type TTG (L/L), at 67.86% (57/84) and 74.29% (52/70), respectively. In Pingqiao and Xiangfu, higher frequencies of the L1014F allele and lower frequencies of the L1014C allele were observed in mosquitoes resistant for beta-cyfluthrin than in those which were sensitive for this insecticide (P< 0.05). The results of Tajima'sDand of Fu and Li'sDandFwere not significantly negative (P> 0.10), and each haplotype was interlaced and did not form two distinct branches.ConclusionsHigh resistance to pyrethroids and propoxur was observed at four sites, but the resistance to malathion varied according to the location.Anopheles belenraeand the L1014W (TGG) mutation inAn. sinensiswere first discovered in Henan Province. The deltamethrin-resistant and deltamethrin-sensitive mosquito populations showed no genetic differentiation. The generation of resistance might be the result of the combination of multiple factors.Graphical AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s13071-023-05796-z.