BACKGROUND: Current guidelines recommend p16 immunohistochemistry (IHC) for testing human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (OPSCC). The need for additional HPV-DNA testing is debated. AIMS/OBJECTIVES: We evaluated the prognostic value of HPV-DNA MATERIAL AND METHODS: A population-based cohort of 1,033 head and neck squamous cell carcinoma patients was used, to identify 124 OPSCC patients treated with a curative intent. RESULTS: Of the p16-positive patients, 17.9% did not show evidence for presence of HPV-DNA in ISH. Both p16 and HPV-DNA ISH were significant prognostic factors for overall survival (OS), disease-specific survival (DSS) and disease-free survival (DFS) when used independently. When combining information from p16 IHC and HPV-DNA ISH, survival of the p16+/ISH- discordant patients was intermediate compared to the p16+/ISH+ and p16-/ISH- groups. In the discordant group, smoking was a strong prognostic determinator, with non-smokers having an outstanding prognosis. CONCLUSIONS AND SIGNIFICANCE: Both p16 IHC and HPV-DNA ISH perform well as separate prognostic biomarkers for OS, DSS, and DFS on a population level. However, a discordant group does exist. Limiting HPV-DNA testing for p16-positive patients who smoke by the time of diagnosis may be a cost-efficient and justified solution.