Human immunodeficiency virus (HIV) infection substantially elevates diffuse large B-cell lymphoma (DLBCL) risk, but its impact on the distinct DLBCL subtypes defined by cell of origin is unclear. We compared DLBCL molecular characteristics and prognosis in 51 HIV-infected and 116 HIV-uninfected cases diagnosed during 1977-2003. Using immunohistochemistry to classify cell of origin based on the Tally algorithm, activated B-cell (ABC)-DLBCL was substantially more common in HIV-infected (83%) than in HIV-uninfected (54%) cases (p < 0.001). Epstein-Barr virus (EBV) was detected in 63% of DLBCLs in HIV-infected cases, occurring almost exclusively in ABC-DLBCL (74% vs. 13% of germinal center B-cell [GCB]-DLBCL, p = 0.002), but was rarely detected in DLBCLs among HIV-uninfected cases (3%). Among HIV-uninfected cases, MYC/IgH [t(8;14)(q24;q32)] and IgH/BCL2 [t(14;18)(q32;q21)] translocations were significantly more common and BCL6/IgH [t(3;14)(q27;q32)] significantly less common in GCB-DLBCL than in ABC-DLBCL (p = 0.010, < 0.001 and = 0.039, respectively). Among HIV-infected cases, translocations other than MYC/IgH [t(8;14)(q24;q32)] (21%) were rare (≤ 6%) and unrelated to cell of origin. ABC-DLBCL was associated with adverse overall survival compared with GCB-DLBCL regardless of HIV status (pHIV-infected = 0.066; pHIV-uninfected = 0.038). Our data demonstrate key differences in the molecular characteristics, cell of origin and prognosis of DLBCL by HIV status in the pre-highly active antiretroviral therapy (HAART) and pre-rituximab era, supporting biologic differences in lymphomagenesis in the presence of HIV.