Background: The association of Epstein-Barr virus (EBV) with Burkitt′s lymphoma (BL) is variable in different geographic regions. In developing countries, the association of EBV with BL is regarded to be of an endemic-type in equatorial Africa ( > 95%) and sporadic-type in the developed countries (15-30%). The purpose of this study is to assess the frequency of EBV infection in BL, in Iran. The study also aims to compare Ribonucleic acid (RNA) in situ hybridization (RISH), the standard diagnostic method, with the polymerase chain reaction (PCR)-based method for diagnosing BL. Materials and Methods: In this epidemiological study, the paraffinized specimens of 18 cases of BL were selected. Next, the ISH of EBV-encoded RNA (EBER-RISH) and PCR assays that were based on Epstein Barr Nuclear Antigen 2 ( EBNA2) amplification were used. The EBV strain was determined by PCR. The data were analyzed using the SPSS10 software and by performing Pearson correlation coefficient formula at a significant level of 0.05. Results: EBV RNA was detected in 50% of the BL specimens. Type 1 and 2 accounted for 70 and 30% of the cases, respectively. Regarding RISH as the standard method for EBV diagnosis, the PCR assays showed a sensitivity and specificity of 100 and 88.9%, respectively. Conclusion: According to the obtained findings, the frequency of EBV in BL was 50% and PCR and RISH showed high concordance and sensitivity in EBV detection. Therefore, PCR can be used as a faster method for EBV detection in high-risk geographical regions.