Objective To investigate the resistance to pyrethroid insecticides and metabolic enzyme levels in Aedes albopictus from Anshun City, Guizhou Province, and to analyze the genotypes and distribution characteristics of the knockdown resistance (kdr) gene, so as to provide a theoretical basis for the chemical control of Ae. albopictus. Methods The larvae of Ae. albopictus were captured from small water containers in residential areas at different locations of Anshun City from July 2023 to December 2024, and then reared in laboratory to F1 to F2 generations. The resistance to deltamethrin, beta-cypermethrin and permethrin was tested in late 3rd-instar and early 4th-instar mosquito larvae of the susceptible strain and field strain with the larval dipping method, and the resistance to the three pyrethroid insecticides was determined in non-blood-fed female adults of the field strain 3 to 5 days following emergence with the tube-test method. The activity of non-specific esterase (NSE), mixed function oxidase (MFO), and glutathione-S-transferase (GST) was measured with a microplate reader in larval mosquitoes without bioassay. Genomic DNA was extracted from individual adult mosquito after bioassay, and partial fragments of the voltage-gated sodium channel (vgsc) gene were amplified using PCR assay. Following sequencing, the sequences were aligned with reference sequences from NCBI to analyze the mutations and frequency distributions of loci V1016, I1532, F1534 and D1763, respectively. The degree of resistance to pyrethroid insecticides in larval mosquitoes was graded using the software SPSS 31.0 professional, and Shapiro-Wilk test and Wilcoxon rank-sum test were performed to determine the distribution of enzyme activity. In addition, the relative importance of metabolic mechanisms and kdr mechanisms in insecticide resistance in Ae. albopictus were predicted by the classification and regression trees (CART) algorithm. Results Larvae of Ae. albopictus exhibited resistance ratios of 38.47, 18.21 and 209.17-fold to deltamethrin, beta-cypermethrin and permethrin, respectively, all appearing a moderate to high resistance level. The mortality rates of adult mosquitoes were 2.04% (2/98), 2.06% (2/97)and 7.00% (7/100), respectively, indicating resistance to all three pyrethroid insecticides. The activities of NSE, MFO, and GST were 356.17 (321.65 to 497.79) nmol α-naphthol/(min·mg pr), 12.88 (11.17 to 18.01) nmol cyt c/mg pr, and 128.70 (116.63 to 153.03) nmol/(min·mg pr) in the field population of Ae. albopictus, respectively, all showing a positively skewed distribution, which were 1.26, 0.45, and 1.65 times higher than those in susceptible strains (Z = ‒4.030, ‒8.191 and ‒7.371, all P values < 0.01). kdr gene mutations were not detected at the locus 1763 in Ae. albopictus from Anshun City, and were detected at loci V1016, I1532, and F1534. At loci 1016 and 1532, wild-type homozygotes were predominant genotypes (65.97%, 98.61%), followed by wild-type/mutant heterozygotes (30.9%, 1.39%), with the lowest proportion seen for the mutant homozygotes (3.13%, 0). There were three alleles at locus 1534, including wild-type (TTC/F), mutant (TCC/S) and mutant (TGC/C), and the mutant-type (TCC/S) had a higher gene frequency than the wild-type (TTC/F). At locus 1534, the mutant homozygote (S/S) had the highest genotype frequency (61.11%), followed by the wild/mutant-type heterozygote (F/S) (28.47%) and wild-type homozygote (F/F) (7.64%), and the mutant homozygote (C/C) had the lowest genotype frequency (2.78%). A total of 10 genotype combinations were detected in the three-locus mutation test, and the T1 genotype (V/V + I/I + S/S) accounted for the highest proportion (60.76%, 175/288), followed by the T2 genotype (V/G + I/I + F/S) (25.69%, 74/288). Results from CART analysis showed that the factors contributing to pyrethroid resistance in Ae. albopictus in Anshun City included mutation at locus 1534 (100%), MFO activity (96.7%), GST activity (66.6%), NSE activity (21.4%), mutation at locus 1016 (16.4%), and mutation at locus 1532 (1.2%). Conclusion Ae. albopictus has developed resistance to pyrethroid insecticides at varying degrees in Anshun City. The NSE, MFO or GST activities have not yet presented typical characteristics of metabolic resistance, and kdr gene mutation is dominated at locus F1534. Emergency of resistance to pyrethroid insecticides in Ae. albopictus results from the synergistic effect of changes in metabolic detoxification enzyme activities and gene mutations.