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To determine rates of anal dysplasia in a cohort of HIV-infected men who have sex with men (MSM), women, and heterosexual men with abnormal anal cytology.We evaluated histologic findings in 728 HIV-infected MSM, women, and heterosexual men referred for high-resolution anoscopy (HRA) after abnormal anal cytology in a single-center cohort study. Using multivariable logistic regression, we evaluated predictors of high-grade squamous intraepithelial lesion (HSIL) histology or invasive carcinoma including age, sexual behavior, receptive anal intercourse (RAI), anogenital warts, smoking status, antiretroviral therapy, CD4+ T-cell count, and HIV-1 plasma viral load.A total of 2075 HIV-positive patients were screened with anal cytology and 62% of MSM, 42% of women, and 29% of heterosexual men had abnormal findings (P <0.001). Of the 728 HIV-infected patients with abnormal anal cytology who underwent HRA, 71% were MSM, 23% women, and 6% heterosexual men. HSIL/cancer was found in 32% of MSM, 26% of women, and 23% of heterosexual men (P = 0.3). There were five cases of anal squamous cell carcinoma (0.7%), four in MSM and one in a heterosexual man. In a multivariable adjusted analysis, biopsy-proven HSIL/cancer was associated with RAI [odds ratio (OR) 2.2; 95% confidence interval (CI) 1.3–3.7]. CD4+ T-cell counts more than 500/μl conferred a lower risk of HSIL/cancer (OR 0.5; 95% CI 0.3–0.9).Rates of anal HSIL histology are high in HIV-infected patients of all sexual risk groups with abnormal anal cytology. Consequently, all HIV-infected patients may warrant anal cancer screening.