Importance: The programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) pathway play an important immunosuppressive role in cancer and chronic viral infection, and have been effectively targeted in cancer therapy. Anal squamous cell carcinoma (SCC) is associated with both human papillomavirus and HIV infection. To date, patients with HIV have been excluded from most trials of immune checkpoint blocking agents, such as anti-PD-1 and anti-PD-L1, because it was assumed that their antitumor immunity was compromised compared with immunocompetent patients. Objective: To compare the local tumor immune microenvironment (TME) in anal SCCs from HIV-positive and HIV-negative patients. Design, Setting, and Participants: Anal SCC tumor specimens derived from the AIDS and Cancer Specimen Resource (National Cancer Institute) and Johns Hopkins Hospital included specimens. Tumors were subjected to immunohistochemical analysis for immune checkpoints (PD-L1, PD-1, LAG-3) and immune cell (IC) subsets (CD3, CD4, CD8, CD68). Expression profiling for immune-related genes was performed on select HIV-positive and HIV- negative cases in PD-L1+ tumor areas associated with ICs. Main Outcomes and Measures: Programmed death-ligand 1 expression on tumor cells and ICs, PD-L1 patterns (adaptive vs constitutive), degree of IC infiltration, quantified densities of IC subsets, and gene expression profiles in anal SCCs from HIV-positive vs HIV-negative patients. Results: Approximately half of 40 tumor specimens from 23 HIV-positive and 17 HIV-negative patients (29 men and 11 women; mean [SD] age, 51 [9.9] years) demonstrated tumor cell PD-L1 expression, regardless of HIV status. Median IC densities were not significantly decreased in HIV- associated tumors for any cellular subset studied. Both adaptive (IC- associated) and constitutive PD-L1 expression patterns were observed. Immune cell PD-L1 expression correlated with increasing intensity of IC infiltration (r = 0.52; 95% CI, 0.26-0.78; P textless .001) and with CD8+ T-cell density (r = 0.35; 95% CI, 0.11-0.59; P = .03). Gene expression profiling revealed comparable levels of IFNG in the TME of both HIV-positive and HIV-negative patients. A significant increase in IL18 expression levels was observed in HIV-associated anal SCCs (fold change, 12.69; P textless .001). Conclusions and Relevance: HIV status does not correlate with the degree or composition of IC infiltration or PD-L1 expression in anal SCC. These findings demonstrate an immune-reactive TME in anal SCCs from HIV- positive patients and support clinical investigations of PD-1/PD-L1 checkpoint blockade in anal SCC, irrespective of patient HIV status.