EBV BamHI fragment H rightward open reading frame 1 (BHRF1), the Epstein-Barr virus (EBV) early gene, is structurally and functionally homologous to the oncogene bcl-2 and may play an important role in the development of EBV-associated tumors. To characterize the polymorphisms of BHRF1 in EBV-associated tumors, we analyzed the sequences of BHRF1 in isolates from nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma (EBVaGC) biopsies as well as throat washing (TW) samples from healthy donors. BHRF1 DNA sequences were analyzed by polymerase chain reaction (PCR) and sequencing for 39 NPC samples, 40 EBVaGC samples, and 53 EBV-positive TW samples from healthy donors. The variants of BHRF1 gene were classified according to the signature changes. The EBV types 1 and 2 at nuclear antigen (EBNA) 3C locus were determined by PCR. Compared with EBV standard cell line B95-8, all isolates carried a silent mutation at amino acid (AA) 80 (nucleotide 54616 T→C), the AA88 L→V mutation was found in most isolates, and the AA79 V→L mutation in a few isolates. Other mutations were sporadically distributed. Based on the mutations at AA88 and AA79, 3 distinct variants of BHRF1 genes, designated as 79V88V, 79L88L, and 79V88L, were identified. The 79V88V was the most common variant. The distribution of the BHRF1 variants among the NPC, EBVaGC, and TW samples was not significant. The corresponding regions of bcl-2 homologues were conserved in all isolates except for 3 samples. The distribution of BHRF1 variants in type 1 and type 2 strains was significant different (P < 0.001, contingency coefficient was 0.554). The 79V88V is the dominant variant in NPC, EBVaGC, and TW samples from healthy donors and preferential linkages between BHRF1 and EBNA3C variants exist. Conserved BHRF1 in Bcl-2 homologous domains is helpful to remain the important role of BHRF1.