We evaluated the usefulness of prognostic markers in patients with diffuse large B-cell lymphoma (DLBCL) treated with cyclophosphamide, vincristine, doxorubicin, and prednisolone (CHOP) +/- rituximab (R-CHOP) in Japan. We studied 730 patients with DLBCL; 451 received CHOP and 279 R-CHOP. We analyzed biopsy samples immunohistochemically for markers of germinal center B cells (CD10, Bcl-6), postgerminal center B cells (Multiple myeloma-1), and apoptosis (Bcl-2). The median follow-up period for surviving patients was 56.4 months for the CHOP group and 25.2 months for the R-CHOP group. DLBCL were categorized as germinal center B (GCB) subtype (352/730; 48.2%) or non-GCB subtype (378/730; 51.8%). In the CHOP group, the high expression of CD10 (P = 0.022) or Bcl-6 (P = 0.021), or GCB subtype (P = 0.05) was associated with better overall survival, whereas the high expression of Bcl-2 (P = 0.001) or MUM1 (P = 0.011), or non-GCB subtype (P = 0.05) was associated with worse overall survival. In the R-CHOP group, however, these biomarkers except Bcl-6 were not significant prognostic factors. The patients with non-GCB subtype showed improved survival in the R-CHOP group (P = 0.756). The International Prognostic Index was a useful clinical marker of survival in the CHOP group (P < 0.001) and also in the R-CHOP group (P < 0.001). Results of improved survival with rituximab addition indicate that the relevance of previously recognized prognostic factors should be re-evaluated.