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Background and purpose: Breast cancer is one of the most common malignancies in women and early diagnosis of this cancer is a key element for successful treatment. Breast cancer is a multistep disease in which a virus can play a role. Epstein-Barr Virus (EBV) is identified as an important factor in human cancer. This study investigated the relationship between EBV and breast cancer. Materials and methods: Sixty seven paraffin-embedded tissue blocks of breast cancer were used and 53 paraffin blocks of non-cancerous breast tissue (26 fibroadenoma and 27 fibrocystic) were collected as the controls. DNA was extracted using the Roche kit, then all samples were analyzed for the presence of beta-globin gene and suitable samples were evaluated for the existence of DNA-EBV through Polymerase Chain Reaction (PCR). X2 test of homogeneity and SPSS ver.15 were used to analyze the data. Results: Two cases negative for beta-globin gene in paraffin-embedded tissue blocks of breast cancer were excluded from the study. EBV was detected by PCR in 23 (35.38%) cases of breast cancer specimens and in 11 (20.75%) cases among the control samples (7 fibroadenoma and 4 fibrocystic). From the total of 118 samples 34 cases (81.28%) were found positive. Conclusion: This study found significant correlation between EBV infection and breast cancer. Further epidemiological, biological and molecular assessments are needed to confirm the possible association between this virus and the process of carcinogenesis.