Vulvar Intraepithelial Neoplasia (VIN) is the precursor lesion of Vulvar Squamous Cell Carcinoma (VSCC), and the differentiated type (dVIN) is more frequently observed in relation to VSCC. In contrast to usual-type VIN (uVIN), which is related to infection by human papillomavirus (HPV), a germline mutation in the p53 gene is thought to be associated with ~90% of dVIN cases. To date, no infectious agent has been identified in association with dVIN, and studies investigating this possibility have been hindered by the difficulty in accurately diagnosing dVIN from small biopsies. Here, we used immunostaining for p16ink4a), a biomarker for HPV infection, to study 14 uVIN high-grade VIN and 14 dVIN cases, and to select 10 dVIN cases to broadly screen for all kn(own viruses using a pan-viral microarray platform (ViroChip). All of the uVIN tissue samples, including 8 warty and 6 basaloid cases, showed positivity with the p16(ink4a) immunostain. The staining pattern was full-thickness for all except two cases in which positive staining was localized in the lower 1/3 of the epidermis. In contrast, immunostaining for p16(ink4a) was negative in all dVIN cases. ViroChip analysis of 10 pure dVIN samples confirmed the absence of human papillomavirus subtypes or any other virus with the exception of a single sample that showed a weak microarray signature to a porcine herpesvirus. Follow-up PCR testing of the sample was negative for herpesvirus, and in-depth metagenomic next-generation sequencing revealed only sequences corresponding to non-pathogenic viral flora and bacterial contamination. In this study, we demonstrated lack of a virus association in 10 dVIN cases. Alternative pathways for carcinogenesis such as the p53 mutation should be considered for investigation of potential treatment options in dVIN.