The objectives of this study were to characterise and compare non-volatile metabolite profiles of raw milk from healthy, subclinical and clinical mastitis cows using a non-targeted proton nuclear magnetic resonance (1 H-NMR) metabolomic approach. A total of 46 metabolites were identified. Multivariate analysis allowed discrimination of milk from healthy and mastitis cows according to their 1 H-NMR metabolite profiles. Significant increases in acetate, formate, lactate, benzoate, hippurate, b-hydroxybutyrate, valerate, alanine, histidine, isoleucine, leucine, N-acetylamino acid, phenylalanine, threonine and valine content were associated with extremely high somatic cell count and clinical mastitis status of the cows. Furthermore, significant rises in hippurate, valerate, N-acetylglucosamine, histidine, isoleucine and leucine content could be used as potential biomarker for indicating subclinical mastitis infection of the udders. Beside the existing biomarkers for diagnosing mastitis in literatures, we found additional candidate metabolites, such as alanine, valerate and N-acetylglucosamine, in correlation with mastitis status of Thai crossbred Holstein dairy cows.