This study is aimed to investigate the role of paired boxed gene 1 (PAX1) methylation analysis by methylationsensitivehigh-resolution melting (MS-HRM) in the detection of high grade lesions in atypical squamous cellscannot exclude high-grade squamous intraepithelial lesion (ASC-H) and compared its performance with theHybrid Capture 2 (HC2) human papillomavirus (HPV) test. In our study, 130 cases with a diagnosis of ASC-Hfrom the cervical cytological screening by Thinprep cytologic test (TCT) technique were selected for triage.Their cervical scrapings were collected and evaluated by using PAX1 methylation analysis (MS-HRM) andhigh-risk HPV DNA test (HC2), followed by colposcopy and cervical biopsy. Chi-square test were used to testthe differences of PAX1 methylation or HPV infection between groups. In the detection of CIN2+, the sensitivity,specificity, the PPV, NPV and the accuracy of PAX1 MS-HRM assay and high-risk HPV (HR-HPV) tests wererespectively 80.6% vs 67.7%, 94.9% vs 54.5%, 83.3%, vs 31.8%, 94.0% vs 84.4%, and 91.5% vs 57.7%. ThePAX1 MS-HRM assay proved superior to HR-HPV testing in the detection of high grade lesions (CIN2+) inASC-H. This approach could screen out the majority of high grade lesion cases of ASC-H, and thus could reducethe referral rate to colposcopy.