Epigenetic alterations are essential in the development of cancers, while epigenome-wide exploration in cervical cancer has been limited. In this epigenome-wide association study (EWAS) we explore differential DNA methylation signatures associated with CIN (cervical intraepithelial neoplasia) grade 3 and cervical cancer to better understand potential drivers and biomarkers of cervical carcinogenesis. 247 women were recruited between 2014 and 2020 (N = 119 benign, N = 74 CIN3/CGIN [cervical glandular intraepithelial neoplasia] and N = 54 cancer). Methylation signatures were obtained from exfoliated cervical cells and sequenced using the Illumina 850 k array. Logistic regression and conditional analyses were used to test for independent associations between Cytosine-phosphate-Guanine (CpG) sites and case-control status, with adjustment for batch, chip, age, and human papillomavirus (HPV) status. 409 CpG sites were strongly associated with CIN3/cancer (p-value <
5 × 10