ZFX gene which encodes Zinc Finger Protein of X chromosome and homologous to ZFY gene of Y chromosome, is considered to be a highly conserved gene in vertebrates and has a great potential in assessing the genetic diversity and differentiation among different species and within their respective breeds.  In this pioneering study on buffalo, we investigated the genetic variation in ZFX gene exon 5 in Nili-Ravi and Kundi buffalo breeds of Pakistan and made comparison with other bovid. We sequenced this gene in 50 animals (25 of each breed). The sequence alignment revealed that 153 bp long nucleotide sequence of buffalo’s ZFX exon 5 gene was homogenous to that of Bos taurus and Ovis aries with 99% homology. Nucleotide variation was observed at position 77 and 131 in Ovis aries and Bos taurus respectively. Both the SNPs were found to be synonymous. Phylogenetic analysis was also carried out using MEGA 6.1 with Bos taurus and Ovis aries along with other reported species. We conclude that the ZFX gene is highly conserved in bovine species. Moreover, ZFX can be very helpful to explicit the relatedness between different buffalo breeds and other bovids.