Primary invasive squamous cell carcinoma of the vagina is rare, and the role of human papilloma virus in its pathogenesis remains unclear. The aims of our study were to determine the distribution of human papilloma virus genotypes in 21 cases of primary invasive squamous cell carcinoma of the vagina and to correlate human papilloma virus genotype with histological subtypes. Patients' clinical records were reviewed for demographic data and the stage of the disease. Tumors (n = 21) were classified according to the World Health Organization criteria. Human papilloma virus genotyping (INNO-LiPA HPV Genotyping) was performed in the whole series, and statistical analysis was performed with Fisher's Exact Test and with Student's t-test. The patients' age ranged from 36 to 88 (mean 65) years. Six cases were keratinizing squamous cell carcinoma, and 15 cases were non-keratinizing squamous cell carcinoma (seven non-keratinizing not otherwise specified, three basaloid, and five warty types). The median age of patients with keratinizing squamous cell carcinoma was 73.8 years and that of non-keratinizing squamous cell carcinoma patients was 61.5 years (P = 0.08). Human papilloma virus DNA was detected in 17 cases (81%): 13 non-keratinizing squamous cell carcinoma (87%) and four keratinizing squamous cell carcinoma (67%) (P = 0.31). The human papilloma virus genotypes identified were: 6, 11, 16, 18, 31, 33, 35, 40, and 58, with human papilloma virus 16 DNA the most prevalent (33%). Invasive squamous cell carcinoma of the vagina is frequently associated with human papilloma virus infection, and human papilloma virus 16 is the most common genotype. Although without statistical significance, keratinizing squamous cell carcinoma is more frequent in older patients, whereas non-keratinizing squamous cell carcinoma more frequently affects younger women. All studied histological subtypes are strongly associated with human papilloma virus infection.