Cytochrome b gene markers have been proved as an efficient and powerful tool for breed characterization and species identification of buffaloes. This study represents the substantial analysis of mitochondrial DNA variation in Pakistani buffalo breeds and provides information about their genetic diversity. In this study partial amplification of cytochrome b gene of 1,061 bp was done and sequencing results showed ten haplotypes. Comparing all fifty samples from two buffalo breeds of Pakistan, fifteen polymorphic sites were observed out of which, twelve codons 42, 71, 118, 120, 199, 235, 269, 297, 318, 327, 350, 355 of mitochondrial cytochrome b gene are monomorphic which translate same amino acids as in the reference protein sequence due to silent mutation while different in DNA sequence. Similarly three codons 163, 246, 337 of mitochondrial cytochrome b are polymorphic and different from the reference sequence with respect to DNA as well as protein sequence. For the further confirmation a panel of nine microsatellite markers was used with high polymorphism information content (PIC). The frequency distribution of these alleles varies from three to eight allele at locus CSSM66 and ILST029 respectively. The results obtained from this study may contribute to the establishment of routine genotyping service of buffalo breeds for buffalo farmers for animal forensic application in case of any dispute. Additionally this study may help for breed characterization and phylogeny of aforementioned breeds of buffalo.