Organic milk has a high risk of food fraud as it can easily be adulterated with non-organic milk. This study aimed to identify metabolite markers for assessing the authenticity of organic milk from Jersey and Yak. In the untargeted strategy, ultra-high performance liquid chromatography-Q Exactive HF-X mass spectrometer coupled with chemometrics analysis was used to screen and identify tentative markers of organic milk from Jersey and Yak. In the targeted strategy, a quick and easy method of ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) was developed to quantify three markers. The peptide of Thr-Ala-Val and D-biotin were determined to be metabolite markers for distinguishing organic and non-organic Jersey milk, whereas trimethylamine N-oxide was determined to be a metabolite marker for distinguishing organic and non-organic Yak milk. These findings provide critical information to facilitate assessments of organic milk authenticity.