Detection of cervical high-grade squamous intraepithelial lesions (HSIL) in patients with equivocal cytological abnormalities, such as atypical squamous cells (ASC) of undetermined significance (ASCUS) or inability to exclude high-grade squamous intraepithelial lesions (ASC-H) is still a challenge. This study tested the efficacy of PAX1 methylation analysis in the triage of cervical ASCUS and ASC-H and compared its performance with Hybrid Capture 2 (HC2) HPV test. A hospital-based case–control study was conducted. Cervical scrapings from patients with ASCUS or ASC-H were used for the quantitative methylation analysis of PAX1 methylation by MethyLight and HPV testing by HC2. Patients with ASC-H or ASCUS with repeated abnormal smears underwent colposcopic biopsy and subsequent therapies. Diagnoses were made by histopathology at a follow-up of 2 years. The efficacies of detecting high-grade lesions were compared. Fifty-eight cervical scrapings with cytological diagnosis of ASCUS (n = 41) and ASC-H (n = 17) were analyzed. One of the 41 (2.4%) ASCUS patients and seven of 17 (41.2%) ASC-H patients were confirmed to have HSIL. After dichotomy of the PMR, PAX1 methylation rates were significantly higher in ASC developing HSIL compared with those developing reactive atypia (87.5% vs. 12.5%, P < 0.001). Testing PAX1 methylation in cervical swabs of patients with ASC confers better sensitivity (87.5% vs. 62.5%) and specificity (98.0% vs. 86.0%) than HC2 HPV testing. We show for the first time that PAX1 hypermethylation analysis may be a better choice than HC2 in the triage of ASCUS and ASC-H. Diagn. Cytopathol. 2013. © 2011 Wiley Periodicals, Inc.