Abstract: To investigate the polymorphism of mitochondrial D-loop in water buffalo, the entire mitochondrial D-loop region of 66 water buffaloes of five different groups were analyzed, and 58 mitochondrial haplotypes with 147 polymorphic sites were detected. The haplotypes of swamp buffaloes and river buffaloes formed two different clusters, based on the phylogenetic trees constructed with ML method using bovine D-loop sequences as outgroup. The trees showed that swamp buffaloes and river buffaloes belong to two distinctly different clusters, and two small clusters were observed in the swamp cluster among the three. By median joining network analysis, three different clusters were also founded, one high diverged clusters between swamp and river buffalo, and two little clusters appeared in the whole swamp cluster. The mismatch distribution analysis showed that there might have been two different indepent population expansion events in the evolution history of buffaloes. Pooled with 211 released D-loop sequences of Genbank, and 129 haplotypes with 158 polymorphic sites were detected in 878 bp multi-aligned region. The phylogenetic tree and median joining network analysis also showed that there were three clusters in all sampled buffaloes, two swamp buffalo’s clusters and one high diverged river swamps. Two obviously wave crests appeared in the mismatch distribution analysis, which meaned that there might have been two different population expanding events in water buffaloes. Based on the above results, it could be predicted that river and swamp buffaloes decented from two different domestication events, and there were at least two ancestor gene pools in the domestication of swamp buffaloes.