The aim of the present study was to identify novel methylation markers for cervical cancer screening and to test the clinical application of the most promising biomarker in cervical scrapings. Methylated-CpG island recovery assay-based microarray analysis was carried out on a discovery set consisting of cervical cancer tissue and normal cervical tissue to identify significantly hypermethylated genes. Five hundred and four CpG islands, corresponding to 378 genes, were differentially methylated between cervical cancer tissue and normal cervical tissue. Among them, 30 genes were significantly hypermethylated. Of the 30 genes, SOX9, PKLR and DLX4 were selected for further validation by direct bisulfite sequencing. The SOX9 gene revealed complete methylation in the cervical cancer tissue and complete non-methylation in the normal control tissue. A TaqMan-based real-time PCR assay was performed to detect the methylation levels of the SOX9 gene in 156 cervical scrapings, including 48 normal cervical scrapings, 30 scrapings with cervical intraepithelial neoplasia 1 (CIN1), 30 scrapings with CIN2-3 and 48 scrapings with squamous cell carcinoma (SCC). The methylation levels (methylation score) of the SOX9 gene increased significantly with the severity of cervical squamous lesions. The area under the receiver operating characteristic (ROC) curve (AUC) revealed that the methylation score of the SOX9 gene could be used to segregate SCC/CIN2-3 from CIN1/normal (AUC, 0.961; p=0.000). At the optimal cut-off value, a sensitivity of 92.3% and a specificity of 89.7% were obtained. In conclusion, SOX9 methylation is frequently involved in cervical carcinogenesis, and may provide a valuable molecular biomarker for early detection of cervical cancer.