Abstract Background Vigilant management of women with high-risk human papillomavirus (hrHPV) is necessary in cancer screening programs. To this end, we evaluated the performance of S5 (targeting DNA methylation in HPV16, HPV18, HPV31, HPV33, and human gene EPB41L3) to predict cervical intraepithelial neoplasia grade 2 or higher (CIN2+) in a sample of hrHPV-infected women referred to colposcopy in the FRIDA Study, a large screening trial in Mexico. A nested case-control sample with women referred to colposcopy either by atypical squamous cells of undetermined significance or higher (ASCUS+) in cytology and/or positive for HPV types 16 or 18 was tested by S5. Seventy-nine cases of CIN2+ were age-matched to 237 controls without a diagnosis of CIN2+ (<CIN2). DNA from exfoliated cervical cells was bisulfite converted and PCR amplified for S5 targets, and methylation was quantified at specific cytosines by pyrosequencing. Results The S5 classifier separated women with CIN2+ from <CIN2 with a highly significant area under the curve (AUC) of 0.75 (95% CI 0.69–0.82), while AUC for CIN3+ was 0.81 (95% CI 0.74–0.89). To optimize sensitivity and specificity for Mexico, an alternative S5 cutoff of 3.7 was implemented to account for overall higher methylation seen in our already triaged women. All three invasive cancers were detected by methylation or HPV16/18 but none by cytology. Sensitivity of S5 for CIN2+ was 62% (95% CI 50.4–72.7%), specificity was 73% (95% CI 66.9–78.5%), and adjusted PPV was 15.1% (95% CI 12.0–18.3%). In contrast, the crude sensitivity of HPV16/18 detection and cytology were 63.3% (95% CI 51.7–73.9%) and 57.0% (95% CI 45.3–68.1%) respectively; specificity was 29.1% (95% CI 23.4–35.3%) and 62.4% (95% CI 55.9–68.6%) respectively, while adjusted PPV was 6.4% (95% CI 4.9–8.1%) and 10.5% (95% CI 8.0–13.1%), respectively. Methylation testing could reduce colposcopy referrals by 30 to 50% with virtually no loss of sensitivity for CIN2+ and CIN3+. Conclusions S5 testing on hrHPV-positive women significantly increased diagnostic information compared to triage by HPV16/18 plus cytology and appears to have clinical utility as an additional test to substantially lessen burdens on colposcopy. Trial registration The FRIDA Study is registered in ClinicalTrials.gov, number NCT02510027.