Abstract Purpose: Epigenetic host cell changes involved in cervical cancer development following a persistent high-risk human papillomavirus (hrHPV) infection, provide promising markers for the management of hrHPV-positive women. In particular, markers based on DNA methylation of tumor suppressor gene promoters are valuable. These markers ideally identify hrHPV-positive women with precancer (CIN2/3) in need of treatment. Here, we set out to identify biologically relevant methylation markers by genome-wide methylation analysis of both hrHPV-transformed cell lines and cervical tissue specimens. Experimental Design and Results: Genome-wide discovery by next-generation sequencing (NGS) of methyl-binding domain–enriched DNA (MBD-Seq) yielded 20 candidate methylation target genes. Further verification and validation by multiplex-targeted bisulfite NGS and (quantitative) methylation-specific PCR (MSP) resulted in 3 genes (GHSR, SST, and ZIC1) that showed a significant increase in methylation with severity of disease in both tissue specimens and cervical scrapes (P < 0.005). The area under the ROC curve for CIN3 or worse varied between 0.86 and 0.89. Within the group of CIN2/3, methylation levels of all 3 genes increased with duration of lesion existence (P < 0.0005), characterized by duration of preceding hrHPV infection, and were significantly higher in the presence of a 3q gain (P < 0.05) in the corresponding tissue biopsy. Conclusions: By unbiased genome-wide DNA methylation profiling and comprehensive stepwise verification and validation studies using in vitro and patient-derived samples, we identified 3 promising methylation markers (GHSR, SST, and ZIC1) associated with a 3q gain for the detection of cervical (pre)cancer. Clin Cancer Res; 23(14); 3813–22. ©2017 AACR.