Abstract: Objective: To investigate the prediction of paired box gene 1 (PAX1) gene methylation on the development of low-grade squamous intraepithelial lesion (LSIL) and its clinical significance. Methods: A total of 191 patients participated in the study,including 58 normal cervical tissue,53 LSIL,29 high-grade squamous intraepithelial lesion (HSIL),and 51 cervical cancer (CC).PAX1 methylation was detected by methylation-sensitive restriction enzyme combined with real-time quantitative PCR (MSRE-qPCR).Receiver operating characteristic (ROC) curves were constructed to quantify the diagnostic performance of PAX1 hypermethylation in distinguishing HSIL from ≤ LSIL samples. Results: The PAX1 gene showed a significant trend of increased methylation levels with the severity of cervical lesions (P<0.01).The ROC curve showed that MSRE-qPCR quantified PAX1 methylation with an AUC of 0.97 for distinguishing CC,0.87 for HSIL,and 0.88 for ≥HSIL.When the Youden index was maximized,the methylation cutoff △Ct=4.0,the specificity was 94.30%,and the sensitivity was 96.00%.For clinical sample screening for HSIL,PAX1 combined with HR-HPV or TCT improved specificity to 95.70% and 96.20%,higher than HR-HPV (20.30%) or TCT (51.20%),or the combination of HR-HPV and TCT (57.80%). Conclusions: PAX1 hypermethylation is an ideal biomarker to differentiate HSIL from LSIL.