Epstein-Barr virus (EBV), a ubiquitous herpesvirus associated with certain lymphomas and carcinomas, has been identified within the malignant cells of a small proportion of breast tumors. As breast cancer is a very common malignancy in women, a pathogenetic role of EBV for even a subgroup of patients could have important implications for etiology and prevention. Therefore, we attempted to confirm the EBV-breast cancer association by exploring it in a representative case series stratified by characteristics that modify breast cancer risk. We studied a sample of 97 female and 28 male patients identified from a US population-based cancer registry. Patients were selected randomly within age, sex, ethnicity and tumor estrogen-receptor status groups. With their archived tumor tissues, we examined EBV presence using in situ hybridization for the EBER-1 transcript. In the 107 technically adequate specimens, we did not detect this viral transcript in any tumors, including one from a woman who also had an EBER-positive nasopharyngeal carcinoma. Our uniformly negative findings are extremely unlikely to have occurred by chance and cannot be attributed to selective sampling, as our study group included persons at diverse risk for breast cancer. We conclude that the EBV EBER-1 transcript is not commonly expressed in breast cancer, based on a broadly representative case series, though we cannot exclude an association of EBV within a particular population subgroup.