Buffalopox virus (BPXV) is considered to be a close variant of vaccinia virus (VACV), the prototype member of the genus Orthopoxvirus. In the present study, we have analyzed the sequences of H3L, A27L, and D8L gene-homologues of VACV in BPXV to elucidate its genetic relationship to VACV and other orthopoxviruses (OPVs). Products of these genes have been shown to be important in attachment of VACV to host cell surface receptors during viral entry. Additionally, the A27L gene is also responsible for cell fusion during infection, while the H3L gene is required for synthesis of the highly immunogenic major envelope protein p35. Full-length nucleotide sequences of H3L, A27L, and D8L genes of three BPXV isolates were determined by PCR amplification, cloning, and sequencing. The nucleotide (nt) sequence and the deduced amino acid (aa) sequences were compared with published sequences from other members of the genus Orthopoxvirus. Comparative sequence analysis of all the three genes revealed high sequence identity of BPXV isolates with VACV (close to 99% sequence identity) at both the nt and aa level. Phylogenetic analysis based on the deduced aa sequences of the H3L, A27L, and D8L genes also showed that BPXVs are more closely related to VACV than to any of the other OPVs.