Abstract Background Insufficient specificity of the high-risk human papillomavirus (hrHPV) assay in primary cervical cancer screening results in unnecessary referral. Additional assays to triage hrHPV-positive women are needed to improve molecular cervical cancer screening. DNA methylation is a promising biomarker in cervical cancer. We evaluated the clinical performance of potentially methylated genes as a triage assay for hrHPV-positive women. Results We conducted a retrospective hospital-based case–control study in Taiwan. Cervical scrapings were collected before colposcopy for hrHPV testing and quantitative methylation-specific PCR (QMSP) of 16 genes. Five genes, POU4F3, HS3ST2, AJAP1, PAX1, and SOX1, were prioritized for the clinical performance to triage hrHPV-positive women. Two hundred cervical scrapings were randomly classified into a training set (n = 111) and testing set (n = 89). All samples were tested for hrHPV using a Hybrid Capture II (HCII) assay. HrHPV-positive women were subjected to DNA methylation analysis by QMSP. In the training set, the receiver operating characteristic (ROC) curves defined the optimal methylation index (M-index) cutoff values for discriminating CIN3+ from CIN1/normal, which then were applied to the testing set. Among the five genes, POU4F3 revealed the highest area under the ROC curve (AUC) (0.86; 95 % CI, 0.78–0.95) in detecting CIN3+. In the testing set, POU4F3 revealed the best clinical performance in triage of hrHPV-positive women with a sensitivity of 74 % and specificity of 89 % for detecting CIN3+. Conclusions POU4F3 methylation analysis is a potential molecular tool for triage in detecting CIN3+ in hrHPV-positive women. The combined use of broad-spectrum HPV assay and POU4F3 methylation analysis as a new generation of molecular cervical cancer screening warrants further population-based study.