A simple and reliable duplex polymerase chain reaction (duplex-PCR) technique is proposed to identify and differentiate cattle and water buffalo DNA using primers that were tested on mitochondrial DNA (mtDNA) extracted from meat muscle samples. Different levels of autolysis were experimentally produced by putrefaction and heating the samples at various temperatures and conditions to simulate the various meat processing technology. The optimized PCR amplified 113 bp and 152 bp fragment of cyt b gene from mtDNA. This test was successful in detecting up to 1 pg adulteration in cattle-buffalo meat mixture. The test is a valuable tool for meat authentication and screening of cooked, putrefied and mixed samples of cattle and buffalo.