Plants cultivated on the Qinghai-Tibetan Plateau grow in an extremely cold environment and thus are exposed to cold stress. To assess the metabolic processes during cold exposure of Tibetan hulless barley (Hordeum distichon L.), metabolic analyses were conducted on one tolerant (XiLa) and one sensitive (ZangQing) cultivar exposed to six temperatures (24 °C, 12 °C, 5 °C, 0 °C, -5 °C, -8 °C) for 24 h. In total, 770 metabolites were identified, including amino acids and derivatives, carbohydrates, flavonoids, lipids, nucleotides and derivatives, and phenolamides. In principal component analysis, the samples were clearly grouped according to the cultivar, suggesting that the two cultivars have differential responses to cold stress. In cold-grown plants, eight metabolites, including monoacylglycerol (MAG, 18:2), MAG (18:3), deoxyadenosine, 6-methylmercaptopurine, and coniferin, were significantly altered in XiLa, but not in ZangQing when compared to the levels in control plants, and thus, these compounds can be considered as potential biomarkers of exposure to cold stress in hulless barley. Furthermore, differentially altered metabolites between seedlings exposed to -8 °C and those maintained at 24 °C were significantly enriched in glutathione metabolism. The findings of this study will be useful for the development of cultivars with cold stress tolerance.