People living with HIV (PLWH) are at highest risk of anal cancer and will benefit from optimized screening for early disease detection. We compared host DNA methylation markers in high‐grade squamous intraepithelial lesions (HSIL) versus samples negative for intraepithelial lesions (NILM) or low‐grade intraepithelial lesions (LSIL) in PLWH. We recruited PLWH identifying as male aged ≥18 years undergoing high‐resolution anoscopy (HRA) in Seattle, Washington, 2015–2016. Anal brush samples were collected for HPV detection, genotyping, and pyrosequencing methylation (host genes ASCL1, PAX1, FMN2, and ATP10A); clinical data were ed from medical records. We assessed associations between methylation and presence and extent of HSIL using generalized estimating equation logistic regression, adjusting for age, CD4 count and HIV viral load. Marker panels using HPV DNA and methylation were also evaluated to predict prevalent HSIL. We analyzed 125 samples from 85 participants (mean age 50.1; standard deviation 11.0 years). ASCL1 (adjusted odds ratio aOR per 1 unit increase mean percent methylation: 1.07, 95% CI: 1.01–1.13) and FMN2 (aOR per 1 unit increase mean percent methylation: 1.14, 95% CI: 1.08–1.20) methylation were significantly associated with HSIL versus NILM/LSIL. ASCL1 (aOR: 1.06, 95% CI: 1.01–1.11) and FMN2 (aOR: 1.13, 95% CI: 1.08–1.17) methylation were positively associated with increasing HSIL extent. A panel combining methylation (ASCL1 and FMN2) and HPV DNA (HPV16, HPV18, and HPV31) demonstrated best balance of sensitivity (78.2%) and specificity (73.9%) for HSIL detection compared with methylation or HPV alone. Increasing levels of DNA methylation of ASCL1 and FMN2 were positively associated with HSIL detection in PLWH. Host gene methylation testing shows promise for HSIL screening and triage. What's New? People living with HIV are at highest risk of anal cancer and would benefit from optimized screening practices for early disease detection. However, evidence to inform clinical guidelines for anal cancer screening and diagnosis remains sparse. Here, the authors compared host DNA methylation markers in high‐grade squamous intraepithelial lesions versus low‐grade intraepithelial lesions or samples negative for intraepithelial lesions in people living with HIV. They also evaluated marker panels using HPV DNA and methylation to predict prevalent high‐grade squamous intraepithelial lesions. The study supports the promise of host gene methylation testing for high‐grade squamous intraepithelial lesion screening and triage.