© 2018 American Association for Cancer Research. Background: HIV-positive women are at substantial risk of HPV-associated cervical neoplasia caused by high-risk (HR) HPVs. Methylation of the HPV genome is associated with cervical intraepithelial neoplasia grade 3 (CIN3) in HIVnegative women, yet it is unknown whether this holds true for HIV-positive women. Methods: We designed a case–control study within the Women's Interagency HIV Study (WIHS) cohort comparing HIV-positive CIN3 cases (N = 72) to HIV-positive controls without detectable CIN2þ. The unit of analysis and matching was HPV-type infection. Cases with 2HR-HPV types (N=23; 32%) had a separate control for each HR-HPV type. We developed and utilized next-generation sequencing (NGS) methylation assays for 12 different HR-HPVs, focusing on CpG sites in the L1/L2 regions. Results: Significant case–control differences in individual CpG site methylation levels were observed for multiple alpha-9 (HPV16/31/35/58) and alpha-7 HPV (HPV18/39/ 45) types, based on dichotomization of tertile levels (T3 vs. T1 and T2). Analyses combining homologous CpG sites [e. g., HPV16-L1-5608/HPV31-L1-5521/HPV35-L2L1-5570; OR = 7.28; 95% confidence interval (CI): 2.75–19.3], and (e.g., HPV18-L1-7062/HPV45-L1-7066; OR = 6.94; 95% CI: 1.23–39.3) were significant in separate case–control comparisons. In cases with multiple HR-HPVs, we tested and confirmed the hypothesis that one HR-HPV type would have higher methylation than other types detected, consistent with there being a single HR-HPV causally related to a lesion. Conclusions: CIN3 is associated with elevated L1/L2 CpG methylation levels in HIV-positive women. Impact: HPV DNA CpG methylation is a promising triage option in HIV-positive women testing positive for HR-HPV types and provides risk attribution in women with multiple HPV type infections.