The phylogenetic diversity of dissimilatory sulfite reductase (DSR, EC 1.8.99.3) alpha-subunit genes from a deep-sea cold seep was analyzed. Bulk genomic DNA was extracted from the cold seep sediment and used for amplification by polymerase chain reaction (PCR) of DSR alpha-subunit gene. Two sizes of PCR products, 1.4 kb (expected) and 1.3 kb (unexpected), were amplified. Sixteen clones of the 1.4-kb amplicons and 16 clones of 1.3-kb amplicons, a total of 32 clones, were obtained and grouped into operational DSR units (ODUs) based on restriction fragment length polymorphism (RFLP) by digestion with HaeIII and MboI. A total of 14 ODUs, i.e., 5 ODUs from 1.4-kb amplicon clones and 9 ODUs from 1.3-kb amplicon clones, were recovered. About 400 bp of the 5' ends of all the clones was sequenced and validated the RFLP-based ODU grouping. All the 5'-end 400-bp sequences of ODUs, even from the 1.3-kb amplicons, showed the characteristic DSR amino acid sequence motifs. The ODUs from 1.4-kb amplicons were closely related to the delta-Proteobacterial lineage with the DSR genes from epsilon-Proteobacterial epibionts of the hot vent worm Alvinella pompejana. The ODUs from 1.3-kb amplicons were mostly related to the unknown but possibly archaeal lineage. The diversity of the DSR genes may indicate the diversity of sulfate reducers in the seep sediment as well as the complexity of electron donors including methane.