Objective: A substantial proportion of vulvar cancers are caused by high-risk human papillomavirus (hrHPV), but hrHPV prevalence in vulvar cancer has mainly been investigated in smaller studies which did not evaluate time trends. Our aim was to assess hrHPV prevalence in >1300 Danish vulvar cancers diagnosed during 1990–2017, including changes in hrHPV prevalence over time. Methods: In a nationwide pathology register, we identified women diagnosed with vulvar cancer at thirteen hospitals from all Danish regions. Archival tumor tissue was collected from local repositories and, upon pathology review, sent to a central laboratory for HPV testing using INNO-LiPA. We calculated hrHPV prevalence according to time, age and histology, and evaluated the overall and age-specific estimated annual percentage change (EAPC). Results: We included 1308 vulvar cancer cases, with a median age of 72 years at diagnosis. The overall hrHPV prevalence was 52.0% (95% CI: 49.3–54.7). HPV types 16/18 were found in 39.6% of cases, whereas nine-valent HPV (9vHPV) vaccine types 16, 18, 31, 33, 45, 52, and 58 were found in 50.8%. The hrHPV prevalence showed an increasing trend over time, with an EAPC of 0.35% (95% CI: 0.00–0.71). The hrHPV prevalence was higher in younger women throughout the study period, and increasing trends over time were seen in both older (age ≥ 60) and younger (age < 60) women. The hrHPV prevalence was higher in non-keratinizing (71.0%) and warty/basaloid (78.0%) carcinomas than in keratinizing (39.4%) and verrucous (36.4%) carcinomas. Conclusions: Our results indicate that the 9vHPV vaccine could potentially prevent a substantial proportion of vulvar cancers in Denmark.