The evolutionarily conserved serine/threonine kinase, AMP-activated protein kinase (AMPK), functions as a cellular fuel gauge that regulates metabolic pathways in glucose and fatty acid metabolism and protein synthesis. AMPK is a heterotrimeric protein consisting of a catalytic ? and regulatory ? and ? subunits. This task yet remains to be undertaken in buffalo a species contributing to many South Asian economic importance, the genetic resource conservation of different buffalo breeds especially Nili-Ravi, Kundi and Azakhali breeds of Pakistan. Sequence analysis revealed thirty one single nucleotide polymorphisms (SNPs) of buffalo PRKAB1 of which four were found in exons and twenty seven were in introns, all exonic SNPs were nonsynonymous mutation. PRKAB1 gene revealed eight different haplotypes resulting from thirty one polymorphic sites and haplotype frequency distribution of the at codon 150, 274, 281 and 282 were also investigated. This work will provide the genetic data for identification of better energy utilizing animals that produce good amounts of milk and meat under the prevailing circumstances.