Molecular species detection in food has become common in the last 20 years. In this study, PCR technique was applied to differentiate buffalo and cattle meat by PCR-buffalo and PCR-cattle. Common forward primer (F2) was designed base on homology region of cytochrome b gene of buffalo and cattle to reduce expenses. Reverse primer RBU2 (PCR- buffalo) and RC2 (PCR-cattle) were designed base on homology region of cytochrome b gene of buffalo and cattle.Mitochondrial DNA fragments of 763 bp, 839 bp for buffalo and cattle, respectively, were amplified. The PCR-buffalo was detected meat buffalo in the treat mixtures at 80°C to 180°C/15 min but it was not detected buffalo meat percentage less 10 percent in the treatment mixtures at 120oC or 130oC in 30 min. PCR-cattle was detected cattle meat in the treat mixtures at 80oC to 180oC/15 min but it was not detected cattle meat percentage less 10 percent in the treated mixtures at 120oC or 130oC from 15 min to 30 min. The minimum DNA concentration was detected by PCR-buffalo and PCR-cattle was 0.001 ng/ul.