Methylated genes have the potential to provide a new generation of cancer biomarkers. The aim of this study was to investigate: (1) the promoter methylation status of DAPK1, RAR-β2 and MGMT in randomly selected normal cytology of the general female population; (2) the effectiveness of gene methylation in liquid-based cytology to help in the early diagnosis of HSIL; (3) the relationship between HPV infection and gene methylation.Methylation of DAPK1, RAR-β2 and MGMT in 667 cervical samples with 331 cases of abnormal cytology and 336 randomly selected normal cytology controls was detected by methylation-specific PCR and denaturing high-performance liquid chromatography method (MSP-DHPLC). The methylation frequencies of each gene were compared.Methylation frequencies for MGMT, RAR-β2 and DAPK1 in normal cytology were 36.9, 42.0 and 46.7%, respectively. There was a trend toward increasing methylation frequency for any gene with age (p = 0.0133). Among abnormal cytology, there was a trend toward increasing number of methylation of any gene with severity of cytology grade (r = 0.1178, p = 0.0026). Methylation frequencies for MGMT and RAR-β2 among cytology of each grade were significantly different (χ ( 2 ) = 6.8976, p = 0.0086; χ ( 2 ) = 33.2477, p < 0.0001), and methylation frequencies for RAR-β2 in ASC were significantly higher than that in negative cytology (χ ( 2 ) = 8.7128, p = 0.0032). The relationship between MGMT, RAR-β2 and DAPK1 gene methylation and HPV infection was not found.This study reported methylation frequencies for MGMT, RAR-β2 and DAPK1 in normal cytology of the general female population. The combination of MGMT methylation, cytology and HPV infection is preferable for early detection of CIN2+ in cytology samples. There was no relationship between MGMT, RAR-β2 and DAPK1 gene methylation and HPV infection.