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Context.--Detection of high-risk human papillomavirus (HR-HPV) in squamous cell carcinoma is important for classification and prognostication. In situ hybridization (ISH) is a commonly used HR-HPV-specific test that targets viral RNA or DNA. The College of American Pathologists (CAP) provides proficiency testing for laboratories performing HR-HPV iSh. Objective.--To compare the analytical performance of RNA- and DNA-based ISH methods on CAP HR-HPV proficiency tests. Design.--Data from the 2016-2018 CAP HPV ISH proficiency testing surveys were reviewed. These surveys consist of well-characterized samples with known status for HR-HPV, including 1 to 2 copies, 50 to 100 copies, 300 to 500 copies, and no copies of HR-HPV per cell. Results.--Ninety-five participants submitted 1268 survey results from 20 cores. Overall, RNA ISH had a significantly higher percentage of correct responses than DNA ISH: 97.4% (450 of 462) versus 80.6% (650 of 806) (P < .001). This disparity appears to be the consequence of a superior sensitivity of rNa ISH compared to DNA ISH for samples with 1 to 2 and with 50 to 100 copies of HRHPV per cell: 95.2% (120 of 126) versus 53.8% (129 of 240), P < .001, respectively, and 100% (89 of 89) versus 76.3% (119 of 156), P < .001, respectively. Conclusions.--An assessment of CAP HR-HPV proficiency test performance indicates that RNA ISH shows significantly higher accuracy than DNA ISH owing to higher analytical sensitivity of RNA ISH in tumors with low (1-2 copies per cell) to intermediate (50-100 copies per cell) HR-HPV viral copy numbers. These data support the use of RNA over DNA ISH in clinical laboratories that perform HR-HPV testing as part of their testing algorithms. (Arch Pathol Lab Med. 2020;144:344-349; doi: 10.5858/arpa.2019-0093-CP), High-risk human papillomavirus (HR-HPV) causes HPV-positive squamous cell carcinomas (SCCs) at multiple anatomic sites including the oropharynx, anus, and urogenital tracts. The detection of HR-HPV in tissue samples containing SCC [...]