This study analysed buffaloes from north-east India and compared their nuclear and mitochondrial DNA variations with buffaloes of mainland India, China, Mediterranean and South-East Asia. Microsatellite genotypes of 338 buffaloes including 210 from six north-east Indian buffalo populations and three mainland Indian breeds were analysed to evaluate their genetic structure and evolutionary relationships. Phylogenetic analysis and multidimensional scaling plot of pairwise FST revealed the clustering of all swamp-type buffaloes of north-east India with Lower Assamese (significantly hybrid type) buffaloes in one plane and all the mainland river buffaloes in another plane while the upper Assamese buffaloes being distinct from both these clusters. Analysis of mtDNA D-loop region of 530-bp length was performed on 345 sequences belonging to 23 buffalo populations from various geographical regions to establish the phylogeography of Indian water buffalo. The swamp buffaloes of north-east India clustered with both the lineages of Chinese swamp buffalo. Multidimensional scaling display of pairwise FST derived from mitochondrial DNA data showed clustering of upper Assamese, Chilika and Mediterranean buffaloes distinctly from all the other Indian buffalo populations. Median-joining network analysis further confirmed the distinctness and ancestral nature of these buffaloes. The study revealed north-east region of India forming part of the wider hybrid zone of water buffalo that may probably extend from north-east India to South-East Asia.