OBJECTIVES: Human papillomavirus (HPV) mediated oropharyngeal cancer (OPC) incidence has increased dramatically. Accurate oral HPV prevalence data are essential for assessing population epidemiology, vaccination and screening programmes. As oral rinse is the most commonly used primary outcome measure for estimating oral HPV prevalence, we aimed to compare the efficacy of oral rinse against other detection methods. METHODS: The Oromouth study enrolled 945 participants, aged 0-65 years (63% female), undergoing tonsillectomy for non-malignant indications, without OPC diagnoses. Samples from oral rinse, pharyngeal wall, tongue base, tonsil tissue and blood were collected. HPV DNA in oral samples was centrally tested via polymerase chain reaction, and serology samples for HPV antibodies. Statistical analyses assessed prevalence, detection rates and ratios. RESULTS: Combining all four oral collection sites, high-risk (HR)-HPV and any HPV prevalence were 4·3% and 18%, respectively. HR-HPV prevalence was similar between males (4·1%) and females (4·5%). Oral rinse had the highest HR-HPV (4·0%) and any HPV detection rates (16%), but still missed 73% (11/15) identified as HR-HPV-positive by other oral sites. Compared to oral rinse alone, an additional 38% (11/29) HR-HPV infections were picked up by the other three oral collection methods. Addition of serology testing did not result in substantial improvement. CONCLUSIONS: Of the four oral sites, oral rinse demonstrates the highest detection rates by far but has important limitations. These findings should be taken into consideration when estimating oral HPV infection rates, especially for vaccination studies and screening programmes.