Water buffalo has been classified into two types, namely; river and swamp, based upon cytological, morphological, ecological, and more recently mitochondrial DNA sequence differences. Our results on the mtDNA D-loop region and the cytochrome b sequence analyses strongly supported the origin of the domestic buffalo from two different but related subspecies, i.e. Bubalus bubalis bubalis and Bubalus bubalis carabanesis. Given these differences and the present-day distribution of the river and swamp buffalo, we proposed an independent domestication of these two types. Our analysis of mitochondrial DNA from various breeds of river buffalo from Asia and Europe is consistent with the available archaeological information that river buffalo has been domesticated in the Northwestern parts of the Indian subcontinent. It appears that the present day domestic buffalo is an outcome of a complex domestication process involving multiple maternal lineages and continuation gene flow from the wild populations after initial domestication. Analysis of microsatellite loci among Indian rivers breeds revealed differentiation values ranging from 0.75% to 6.00% between various breed combinations and showed mainly four clusters i.e. Toda, Jaffarabadi, and Pandharpuri breeds as one cluster each, and the Bhadawari, Nagpuri, Surati, Mehsana and Murrah breeds as admixture. Clustering of these breeds based upon mtDNA D-loop region mostly matched the differentiation patterns seen from the microsatellite data, indicating a deep time depth of differentiation of maternal lineages in Indian buffalo. Thus, the very concept of ‘breed’ in river buffalo may have a different connotation as compared that in cattle in the Western world.