Alfalfa (Medicago sativa L.) is a very important forage that is severely threatened by water deficiency. Metabolomic changes under drought stress conditions are largely unknown in alfalfa. Therefore, two alfalfa (Medicago sativa L.) cultivars with contrasting tolerance to drought, namely the drought-tolerant Dryland "DT" and drought-sensitive WL343HQ "DS", were investigated to identify metabolic responses to drought stress. Both the tolerant and sensitive alfalfa showed significant differences in physiological levels under water-limiting conditions. The drought-tolerant cultivar DT sustained growth despite reduced water potential and photosynthesis, suggesting that the functioning of distinct metabolic pathways contributed to enhance tolerance. There were more differentially expressed metabolites produced by DT compared to DS under drought conditions. In addition, the drought-tolerant DT cultivar accumulated more tryptophanol, homocarnosine, S-adenosylhomocysteine, cytidine and clairol, which may be important metabolites for alfalfa drought tolerance. In addition, 17-Hydroxymethylethisterone with unknown function was a candidate tolerance metabolite, whereas the drought-sensitive cultivar DS produced reduced levels of lipid metabolites and increased levels of biosynthesis of amino acids. Among them, the up-regulated L-Proline, L-Arginine and L-Leucine were evident, supporting the conclusion that DS suffered more severe drought stress. This study provided new insights into the metabolic changes of alfalfa in drought stress conditions.