Background: The S5-methylation test, as an alternative classifier to cytology and HPV16/18 genotyping to triage cervical squamous intraepithelial lesions, has not been widely validated in Asian countries. Herein, we compared S5 methylation to HPV16/18 and cytology to detect cervical high-grade squamous intraepithelial lesion (HSIL) in a screening population who with either HPV infection or abnormal cytology results or both of them, derived from a multi-central clinical trial of 2246 Chinese participants. We matched all ≥HSIL+ cases (n=468) with ≤LSIL controls (n=468) to analyze the effectiveness of methylation. Methylation of S5 was quantified by pyrosequencing, blinded to cytology, histological and initial HPV results. Results: The S5 methylation could distinguish women with ≥HSIL+ from women with ≤LSIL at a high area under the curve (AUC) of 0.80 (95% CI 0.74-0.85). The sensitivity of S5 methylation (at 2.85 cutoff) for ≥HSIL+ was 76.1% (95% confidence interval [CI] 71.7-79.2) was higher than HPV16/18 sensitivity (64.9%, 95% CI 58.3-71.7, P = 0.039) or cytology (48.9%, 95% CI 42.8-53.2, P < 0.001). At this cutoff, the specificity of S5 for ≥HSIL+ was (79.9%, 95% CI 76.2-84.9), higher than HPV16/18 (44.8%, 95% CI 40.1-49.4, P < 0.001) and cytology (54.6%, 95%CI 50.7-57.9, P < 0.001). In addition, S5 methylation could provide predictive information about progression in specific population in follow-up period. Conclusion: S5 methylation classifier with high sensitivity and specificity exceeded HPV16/18 or cytology for detecting women with ≥HSIL+ in a screening Chinese population with HPV infection and/or abnormal cytology results. Furthermore, S5 methylation is a potential classifier for predicting progression.