Background: Breast cancer is the most common cancer in women worldwide and in Sudan. Breast cancer occurs due to a multifactorial process and infection with an oncogenic virus has been recently investigated as a possible risk factor for breast cancer. For nearly two decades, studies have incriminated Epstein-Barr virus (EBV) in the etiology of breast cancer. However, the results are unconvincing, and their interpretation has remained a matter of debate. The aim of this study was to detect EBV in breast cancer biopsies obtained from Sudanese female patients. Methods: A descriptive, hospital-based, case-control study, conducted at Faculty of Medical Laboratory Science, University of Khartoum, Khartoum, Sudan. Archival blocks were obtained from 115 patients with breast cancer and 115 controls during the period between November 2016 till March 2017. Results: Among 115 breast cancer tissue specimens, EBV DNA was identified in 42/115 (36.5 %) samples and was not identified in 73/115 (63.5 %) tissue samples. The highest frequency of EBV detection was among 41–60 year-olds (23/42, 54.7 %), followed by 21–40 year-olds (12/42, 28.5 %) and 61–80 year-olds (5/42, 11.9 %). In the control group, the majority were diagnosed with fibroadenoma (70.4%), followed by fibrocystic changes (10.4%) and lactating changes (0.9%). Conclusion: The data obtained in this study demonstrated that EBV was present in a high percentage of our study population; however, the exact role of EBV in Sudanese breast cancer needs to be studied more in depth.