BackgroundDengue fever is an infectious disease that is imported into Shanghai, China and requires prevention and control measures. Controlling the vectorAedes albopictusthrough insecticide use is a key approach to dengue control. However, the rapid evolution of insecticide resistance inAe.albopictushas raised concerns about the failure of dengue control efforts. Knockdown resistance (kdr) caused by point mutations in the voltage-gated sodium channel (VGSC) gene is a primary mechanism of pyrethroid resistance. In this study, we investigated thekdrmutations ofAe.albopictusin Shanghai and evaluated the trend in its evolution.Methodology/principal findingsWe collected 17 populations ofAe.albopictusfrom 15 districts in Shanghai in 2020, extracted genomic DNA from individual mosquitoes, and amplified Domain II, III, and IV inVGSCusing PCR. Following sequencing, we obtained 658VGSCsequences. We detected the nonsynonymous mutations V1016G, I1532T, and F1534S/C/I, among which V1016G and F1534C/I were reported in Shanghai for the first time and F1534I was a novel mutant allele inAe.albopictus. The overall mutation frequency was 84.65%, with individual mutation frequencies ranging from 46.81% to 100%, excluding the Fengxian District population, which had a frequency of 0%. The V1016G and I1532T mutation types accounted for 7.14% and 3.42%, respectively. The mutant allele at codon 1534 accounted for 63.98% of all mutations, including TCC/S (62.77%), TGC/C (1.06%), and ATC/I (0.15%). We identified and classified five intron types in Domain III by length, including A (83 bp, 12.07%), B (68 bp, 87.30%), C (80 bp, 0.16%), D (72 bp, 0.16%), and E (70 bp, 0.31%). Individuals with intron B had a significant mutation tendency at codon 1534 relative to intron A (chi-square test,p< 0.0001). We found no correlation between mutation frequency and the amount of pyrethroid used (Pearson correlation,p= 0.4755).Conclusions/significanceIn recent years,kdrmutations in theAe.albopictuspopulation in Shanghai have rapidly evolved, as evidenced by an increase in mutation types and significantly increased mutation frequency. The F1534I/ATC mutant allele was found to be a novel mutation, F1534C/TGC was reported for the first time in Shanghai, and intron B in Domain III was significantly associated with mutation frequency at codon 1534. Continuous monitoring of resistance changes and strict regulation of insecticide use are required.