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The aim of this study was to investigate the methylation status of E2BSs in the HPV 16 long control region (LCR) in clinical cervical samples. Methylation status of the four E2BSs in 43 clinical cervical samples with HPV 16 infection was quantitatively detected using pyrosequencing. Meanwhile, Quantivirus® HPV E6/E7 RNA 3.0 assay (bDNA) was used to detect E6/E7 mRNA levels in the corresponding specimens. Our results showed that methylation status of E2BS1, 2 and 4 sites in high-grade squamous intraepithelial lesions (HSIL) and cervical cancer were significantly higher than that of asymptomatic HPV 16 infection and low-grade squamous intraepithelial lesions (LSIL) (all P <.05). Furthermore, methylation status of HPV 16 E2BS1 and 2 was positively correlated with E6/E7 mRNA levels (r s = 0.529 and 0.512 respectively, P <.01). Receiver operating characteristic curve analysis was used to compare the diagnostic performance of E2BSs methylation. When the Youden index was the maximum value, the methylation level of E2BS1 and E2BS2 all demonstrated optimum diagnostic sensitivity of 77.8%, and specificity of 80% and 92%, respectively. The methylation status of E2BS1 and 2 may have utility as diagnostic markers for the severity of cervical lesions in the future. • This study presents new data on the methylation status at four E2BSs of HPV-16 LCR. • The methylation status of E2BSs may affect transcriptional regulation of E2 on E6/E7. • HPV DNA methylation and the severity of cervical lesions appear to be correlated. • Methylation status of E2BSs may be used as diagnostic biomarker for cervical cancer. [ABSTRACT FROM AUTHOR]