Background: Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma. There are various types of DLBCL including immunoblastic and centroblastic. Epstein-Barr virus (EBV) is a member of Herpes virus family found in all human populations inducing different lymphoproliferative disorders. The role of EBV in the development of DLBCL is known. Multiple laboratory methods are available for detecting EBV. This study was conducted to determine the correlation of EBV with DLBCL in samples referred to pathology ward in Shariati and Sina Hospitals by chromogenic in situ hybridization (CISH) method.Methods: In this case/control study, pathological specimens of 50 patients with DLBCL as well as 50 reactive lymph nodes and tonsils (control group) were collected from archives of Shariati and Sina Hospitals and were evaluated for EBV encoded RNA (EBER) expression based on CISH method. A peptide nucleic acid (PNA) EBV probe (Dakocytomatin) was used while all the processes were done in RNAase-free conditions using RNAase-free water, sterile gloves and samplers. Results: Out of fifty specimens in the case group, eight were positive for EBER in comparison with two in the control group (P=0.046). No statistically significant difference was observed between intranodal or extranodal samples (P=0.736) or between males and females (P=0.0746).Conclusion: Our study showed that EBV positivity for EBER in patient with DLBCL could be determined more effectively by CISH method than immunohistochemistry (IHC). Comparative analysis between CISH, PCR and IHC methods is recommended.