BACKGROUND: The purpose of this study was to examine the prevalence of human papillomavirus (HPV) in patients with head and neck squamous cell carcinoma of unknown primary (CUP). METHODS: All patients diagnosed with and treated for CUP between January 1, 2000, and June 1, 2011, at two Danish medical centers were included. All patients received a thorough diagnostic work-up, including FDG-PET, before being diagnosed as CUP. We determined the HPV status in all patients using a combination of HPV DNA PCR and p16 stain. In addition, clinical information on the study patients was retrieved from clinical records. RESULTS: Of the identified 60 patients with CUP, 13 were shown to be positive for HPV DNA, amounting to 22% of the study population. In addition, we were able to show a clear disease-free and overall-survival benefit in the HPV-positive group, with a hazard ratio of 0.16 (95% CI: 0.038-0.67) for over-all survival. This survival benefit was also apparent when adjusted for advanced age in a multivariate Cox regression analysis. CONCLUSION: A fairly large percentage of CUP cases are HPV-related, and because this is related to both the location and prognosis, we recommend HPV testing as part of the diagnostic work-up.