While the etiology of breast cancer remains enigmatic, some recent reports have examined the role of human papillomavirus (HPV) in breast carcinogenesis. The purpose of this study was to determine the prevalence of HPV in breast cancer tissue using PCR analysis and sequencing. Fifty-four (54) fresh frozen breast cancers samples that were removed from a cohort of breast cancer patients were analyzed. Samples were tested for HPV using comprehensive PCR primers, and in situ hybridization was performed on paraffin embedded tissue sections. Findings were correlated with clinical and pathological characteristics. The HPV DNA prevalence in the breast cancer samples was 50% (27/54) with sequence analysis indicating all cases to be positive for HPV-18 type. While HPV patients were slightly younger, no correlation was noted for menopausal status or family history. HPV positive tumors were smaller with earlier T staging and demonstrated lesser nodal involvement compared to HPV negative cancers. In situ hybridization analyses proved negative. The high proportion of HPV positive breast cancers detected in this series using fresh frozen tissues cannot be dismissed, however the role of HPV in breast carcinogenesis remains unclear and may ultimately be ascertained by monitoring future breast cancer incidence amongst women vaccinated against high risk HPV types. J. Med. Virol. 83:2157–2163, 2011. © 2011 Wiley Periodicals, Inc.