Objectives:Anal cancer risk is elevated in MSM with HIV (MSMWH). Anal high-risk human papillomavirus (hr-HPV) infection is necessary but insufficient to develop high-grade squamous intraepithelial lesion (HSIL), the anal cancer precursor, suggesting additional factors. We sought to determine whether the microbiome of the anal canal is distinct by comparing it with the microbiome of stool. We also sought to determine whether changes in the anal microbiome are associated with HSIL among MSMWH.Design:Cross-sectional comparison of the microbiome of the anal canal with the microbiome of stool in MSMWH and cross-sectional comparison of the anal microbiome of MSMWH with anal HSIL with the anal microbiome of MSMWH without anal HSIL.Methods:Sterile swabs were used to sample the anus of MSMWH for microbiome and HPV testing, followed by high-resolution anoscopy. Stool samples were mailed from home. 16S sequencing was used for bacterial identification. Measures of alpha diversity, beta diversity, and differential abundance analysis were used to compare samples.Results:One hundred sixty-six anal samples and 103 matching stool samples were sequenced. Beta diversity showed clustering of stool and anal samples. Of hr-HPV-positive MSMWH, 31 had HSIL and 13 had no SIL. Comparison of the microbiome between these revealed 28 different species. The highest-fold enrichment among MSMWH/hr-HPV/HSIL included pro-inflammatory and carcinogenic Prevotella, Parasuterella, Hungatella, Sneathia, and Fusobacterium species. The anti-inflammatory Anaerostipes caccae showed the greatest reduction among MSMWH/hr-HPV/HSIL.Conclusion:The anal microbiome is distinct from stool. A pro-inflammatory and carcinogenic environment may be associated with anal HSIL.