BACKGROUND: Detection and treatment of anal histologic high-grade squamous intraepithelial lesions (hHSIL) prevents anal cancer. However, anal hHSIL incidence among women with human immunodeficiency virus (HIV, WHIV) remains unknown. Performance of anal high-risk human papillomavirus ([hr]HPV), anal cytology (anal-cyt), and both for hHSIL detection longitudinally over 2 years also remains undetermined. METHODS: We determined 2-year incidence and cumulative risk estimates (2-y-CR) of anal hHSIL among WHIV using prevalence and incidence (per 100 person-years [py]) observations stratified by baseline hrHPV and/or anal-cyt results. RESULTS: In total, 229 WHIV with complete baseline data were included in the analysis; 114 women without prevalent anal hHSIL were followed with 2 annual evaluations. Median age was 51, 63% were Black, and 23% were Hispanic. Anal hrHPV or abnormal anal-cyt was associated with an increased risk of incident anal hHSIL at 2 years (18.9/100py [95% confidence interval {CI} 11.4-31.3] and 13.4/100py [95% CI 8.0-22.7], respectively) compared with no detection of anal HPV or negative cytology (2.8/100py [95% CI 1.1-7.4] and 4.2 [95% CI, 1.8-10.2]) The presence of anal hrHPV with abnormal cytology was associated with 2-y-CR of anal hHSIL of 65.6% (95% CI 55.4%-75%); negative hrHPV with negative cytology was associated with 2-y-CR of anal hHSIL of 9.2% (95% CI 7.0-16.0). CONCLUSIONS: Detection of anal hrHPV or abnormal anal cytology are comparable predictors for 2-y-CR of anal hHSIL. The absence of anal hrHPV combined with negative cytology was predictive of a lower (but measurable) risk of developing anal hHSIL. These findings provide important data to inform anal cancer screening guidelines for WHIV.