Yak milk contains a greater percentage of protein and has better quality than bovine milk. There has been an increasing focus on yak milk and milk products during the last few years. In the present study, a PCR-based assay was developed for the specific identification of bovine milk in yak milk by designing 3 primers targeting the mitochondrial ND1 gene. The use of 3 primers in a single PCR reaction set yielded 2 amplification fragments of 293 and 190 bp from bovine milk DNA, whereas only 1 amplification fragment of 293 bp was obtained in yak milk DNA. The technique was applied to raw and heat-treated binary mixtures of yak and bovine milks and enabled the specific detection of bovine milk with a detection limit of 0.1%. The assay developed is sensitive, fast, and straightforward, and it might be useful in the quality control of yak milk and milk products.