Objective To analyze and compare the DNA methylation levels of three subspecies of Culex pipiens complex, including Cx. p. pallens, Cx. p. molestus and Cx. p. quinquefasciatus, and the DNA methylation levels of Cx. p. quinquefasciatus before and after blood-feeding. Methods Mosquitoes of the 3 subspecies were collected at 5 days post-feathering without blood-feeding and Cx. p. quinquefasciatus were collected at 3 days after blood-feeding. DNA was extracted and sonicated into fragments of approximately 250 bp. The fragmented DNA was sequenced, and the data were aligned with the reference genome sequence of Cx. p. quinquefasciatus (Taxonomy ID: 7176). Methylation levels of the 3 subspecies were analyzed at the genomic, chromosomal and elemental levels (the methylation level above 3% was considered hypermethylated). The differences in methylation levels among the 3 subspecies mosquitoes without blood-feeding, and in Cx. p. quinquefasciatus before and after blood-feeding were compared. The sites with P < 0.001 and absolute value of methylation difference > 5 were identified as differentially methylated sites. The regions with Q < 0.05 and the absolute value of methylation difference > 3 were identified as differentially methylated regions (DMR). The genes with the nearest transcription start site (TSS) to DMRs were identified as DMR-associated genes, which were subjected to gene ontology (GO) enrichment analysis. Results The genome-wide methylation levels of Cx. p. pallens, Cx. p. molestus and Cx. p. quinquefasciatus were 0.454%-0.672%, 0.491%-0.649% and 0.499%-0.655%, respectively, all were below 3%. The methylation levels of CHH of Cx. p. pallens, Cx. p. molestus and Cx. p. quinquefasciatus were 0.631%, 0.618% and 0.624%, respectively, which were higher than CHG (0.567%, 0.559%, 0.559%) (t = 7.14, 83.43, 6.87, all P < 0.05) and CG/CpG (0.508%, 0.505%, 0.505%) (t = 10.59, 12.52, 13.33, all P < 0.05). There were 56 hypermethylated sites and 11 hypermethylated regions present among all 3 subspecies. No significant differences were found among the 3 subspecies (F = 0.07, P > 0.05) at genome-wide methylation levels. No significant differences were found in methylation levels of chromosome 1 (.568%, 0.562%, 0.565%), 2 (0.573%, 0.564%, 0.566%) and 3 (0.575%, 0.566%, 0.569%) among the 3 subspecies at the chromosome level (F = 0.05, 0.11, 0.13, all P < 0.05) nor in the methylation levels of promoters (0.567%, 0.552%, 0.556%), exons (0.562%, 0.556%, 0.558%), introns (0.561%, 0.550%, 0.555%) and TSS (0.579%, 0.506%, 0.621%) among the 3 subspecies at the chromosomal elements level (F = 0.37, 0.06, 0.06, 0.16, all P > 0.05). There were 178 differentially methylated sites and 4 DMRs between Cx. p. pallens and Cx. p. molestus; 209 differentially methylated sites and 8 DMRs between Cx. p. pallens and Cx. p. quinquefasciatus; and 215 differentially methylated sites and 11 DMRs between Cx. p. molestus and Cx. p. quinquefasciatus. GO enrichment analysis revealed that the DMR-associated genes were mainly enriched in the biological processes with response to radiation, light stimuli and abiotic stimuli. The genome-wide methylation levels of Cx. p. quinquefasciatus slightly increased from 0.602% before blood-feeding to 0.617% after blood-feeding, without statistically significant differences (t = 1.21, P > 0.05). The methylation levels of chromosome 1, 2 and 3 in Cx. p. quinquefasciatus were 0.569%, 0.569% and 0.572% before blood-feeding, and were 0.596%, 0.597% and 0.600% after blood-feeding. There were no statistically significant differences before and after blood-feeding (t = 1.31, 1.33, 1.30, all P > 0.05). The methylation levels of the promoters, exons, introns and TSS in Cx. p. quinquefasciatus before blood-feeding were 0.557%, 0.561%, 0.560%, 0.552%, and were 0.585%, 0.584%, 0.584%, 0.594% after blood-feeding, respectively. There were no statistically significant differences before and after blood-feeding (t = 1.48, 1.35, 1.20, 1.69, all P > 0.05). There were 6 DMRs in Cx. p. quinquefasciatus between before and after blood-feeding. GO enrichment analysis showed that the DMR-associated genes were mainly enriched in endosomes and vesicles in cell components, and protein binding or small GTPases binding in molecular functions. Conclusion The genome-wide methylation levels of Cx. p. pallens, Cx. p. molestus and Cx. p. quinquefasciatus are relatively low. The DMR-associated genes are mainly related to biological processes that respond to abiotic stimuli. The methylation level of Cx. p. quinquefasciatus slightly increases after blood-feeding, and the DMR-associated genes before and after blood-feeding are mainly involved in protein binding.