Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of malignant lymphoma. The incidence of Epstein–Barr virus (EBV)-positive DLBCL in Asian and Latin American countries ranges from 8 to 10%. The prognosis of patients with EBV-positive DLBCL is controversial. To compare the clinical outcome of EBV-positive and EBV-negative patients with DLBCL in the rituximab era, we analyzed 239 patients with de novo DLBCL diagnosed between January 2007 and December 2011. The presence of EBV in lymphoma cells was detected using EBV-encoded RNA in situ hybridization, and it was found that 18 (6.9%) of 260 patients with diagnosed DLBCL tested positive. Among the 260 cases, 216 cases were treated with rituximab plus chemotherapy, as were 8 EBV-positive DLBCL patients. The median overall survival and progression-free survival times in patients with EBV-positive DLBCL were 8.7 months and 6.8 months, respectively. The median overall survival and progression-free survival could not be determined in EBV-negative DLBCL patients (P = 0.0002, P < 0.0001, respectively). The outcome of patients with EBV-positive DLBCL remains poor, even in the rituximab era.