Vulvar cancer is rare, but causes substantial morbidity in affected patients. A subset of vulvar cancers is caused by high-risk human papillomavirus (hrHPV), which primarily exerts its oncogenic effect through upregulation of tumor suppressor protein p16.Tumors positive for both hrHPV and p16 (double positive) are assumed to be HPV-driven, but only few large studies have investigated the combined prevalence of hrHPV and p16 positivity in vulvar cancer over time. In this Danish cross-sectional study, we assessed the prevalence of p16 positivity and double positivity for hrHPV and p16 in a large sample of vulvar squamous cell carcinomas (VSCCs) diagnosed during 1990 to 2017. In a nationwide register, we identified VSCCs from 13 hospitals across Denmark, and collected archival tumor tissue for hrHPV testing with INNO-LiPA and immunohistochemical p16 staining. We calculated the prevalence of hrHPV, p16 positivity and double positivity according to time, age and histological Abbreviations: 4v, four-valent; 9v, nine-valent; EAPC, estimated annual percentage change; FFPE, formalin-fixed paraffin-embedded; H&E, hematoxylin and eosin; HPV, human papillomavirus; hrHPV, high-risk human papillomavirus; IHC, immunohistochemical; NOS, not otherwise specified; pRb, retinoblastoma protein; VSCC, vulvar squamous cell carcinoma.