The domestic water buffalo is a major source of milk, meat, draught, hide and employment to the small farmers in many Asian countries especially Pakistan. The phylogeny and genetic structure of water buffalo (Bubalus bubalis) is still not explored well in Nili, Ravi, Nili-Ravi, Kundi and Azakheli buffalo breeds of Pakistan. The genetic characterization of these breeds were completed using mitochondrial D-loop region and FAO/ISAG recommended microsatellite markers through sequencing and genotyping respectively. A reasonable number of SNPs and Allele variations were identified. The phylogenetic relationships among these breeds as well as with other breeds of the world were constructed using certain Bioinformatics softwares. This revealed very interesting information about phylogeny, history of domestication and origin of water buffalo in Pakistan and added valuable to the AnGR of the region. The work provided the basic data which is very helpful for determining the genetic diversity of buffaloes, breed identification, forensic and paternity tests and designing effective breeding and conservational policies in future.