To study the etiologic factors of non-familial breast cancer, the polymerase chain reaction (PCR) and Southern hybridization were used to detect six viruses including human papillomavirus (HPV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV)-1, HSV-2, and human herpesvirus (HHV)-8 DNA in 69 patients with breast cancer and 60 specimens from non-cancerous or other individuals with thyroid tumors or fibroadenoma (non-breast cancer controls). Two specimens from patients with a familial history of breast cancer and five breast cancer specimens with negative results for beta-globin, which was used as internal control, were excluded from this study. Eight (12.9%) HSV-1, 28 (45.2%) EBV, 47 (75.8%) CMV, 8 (12.9%) HPV, and 28 (45.2%) HHV-8 positive samples out of the 62 breast cancer specimens were detected; no HSV-2 DNA was detected in any group. Among the viral gene-positive breast cancer samples, 12 (23.1%) were positive for 1 virus, 16 (30.8%) were positive for 2 viruses, 21 (40.4%) were positive for 3 viruses, and 3 (5.8%) were positive for 4 viruses. Among the viral gene-positive specimens of the control groups, only one virus, CMV, was found in the non-cancerous and thyroid tumor specimens, while multiple viruses were found in the fibroadenoma specimens. The viruses associated with breast cancer were HHV-8 > EBV (P <0.01). The viruses associated with fibroadenoma were HSV-1 and HHV-8 > EBV (P <0.01). The presence of more than one virus was found predominantly in breast cancer and exclusively found in fibroadenoma. CMV was the only virus associated with thyroid tumors.