Approximately 40% of vulvar squamous cell carcinoma (vSCC) cases are etiologically associated with high-risk human papillomaviruses (HPVs) of the alpha genera (α-HPV) that cause other anogenital cancers; however, the etiology of α-HPV-negative vSCC is poorly understood. HPVs of the beta genera (β-HPV) are risk factors for cutaneous squamous cell carcinoma (cSCC) and may be related to carcinomas originating in other cutaneous sites such as the vulva. In this study, we investigate the presence of β-HPVs, with an emphasis on p16-negative squamous lesions adjacent to vSCC. We subjected 28 vulvar squamous intraepithelial lesions adjacent to vSCC for comprehensive HPV genotyping, p16 and p53 immunohistochemistry, and consensus morphology review. Selected cases were subjected to qPCR and RNA in situ hybridization. Clinical data were obtained from medical records. β-HPV DNA was detected in eight of ten p16-negative lesions and three of fourteen p16-positive high-grade squamous intraepithelial lesions. The HPV DNA loads in vulvar squamous intraepithelial lesions ranged between less than 1 HPV DNA copy per cell to more than 100 HPV DNA copies per cell. This is, to the best of our knowledge, the first report of the association of p16-negative vulvar intraepithelial squamous lesions with detection of β-HPVs. These findings expand possible etiologic mechanisms that may contribute to p16-negative lesions of the vulva.