Zooarchaeological data indicates that the Near East and the Indus Valley were the centers of domestication and diffusion of modern day cattle. To better understand the origin and genetic diversity of native cattle breeds (Bos indicus) in Pakistan, partial sequencing of the mitochondrial gene cytochrome b (339 bp) was done in 136 individuals from 10 different breeds. Analysis of the Cyt b gene showed high conservation in Pakistani cattle as expected, with only 26 individuals showing nucleotide changes. Only 5 point mutations were present in multiple individuals (SNP), but one was specific for indicine cattle. Two Lohani and 5 Nari Master cattle showed nucleotide changes specific to taurine cattle. Of the changes found, only three produced amino acid changes in the protein sequence. The UPGMA tree showed a clear differentiation between taurine and indicine cattle, except mitochondrial taurine sequences in Lohani and Nari Master breeds. The within-breed estimates of divergence were very low in all breeds except for Nari Master (mixed-bred). The estimates of divergence among breeds were also low for most breed pairs, except for Nari Master and Dhanni. While the overall genetic divergence within the B. indicus or within B. taurus were also very low (0.002 and 0.003, respectively), however the genetic difference between B. indicus and B. taurus was significantly higher (0.014 ).