Abstract: A study has been undertaken to investigate the effects of heat stress on the primary metabolites of tall fescue. The heat-tolerant genotype PI 578718 and the heat-sensitive genotype PI 234881 were treated with heat stress (40℃/35℃ for 12 h/12 h) for 21 days in order to determine changes in physiological parameters and metabolites. The results showed that grass quality, chlorophyll content, leaf relative water content, and root activity were all higher in the heat-tolerant PI 578718 than in the heat-sensitive PI 234881, while the value of leaf electrolyte leakage was lower in PI 578718. These results indicate that PI 578718 exhibited significantly more heat tolerance than PI 234881. Polar metabolite analysis of leaf and root using gas chromatography-mass spectrometry identified 25 heat-responsive metabolites in the two tall fescue genotypes, mainly consisting of organic acids, amino acids, sugars and sugar alcohols. Most of these metabolites showed higher accumulations in PI 578718 compared with PI 234881, especially following long-term (21 days) heat stress. The differentially accumulated metabolites, including a sugar alcohol (inositol), two organic acids (citric acid and hexadecanoic acid), one sugar (sucrose) and three amino acids (Val, Glu and Pro), may be positively associated with the differential heat tolerance of the two genotypes. Moreover, citric acid mainly served as an antioxidant and intermediate in respiration metabolisms. Its involvement in other defense pathways may play a vital role in tall fescue adaptation to heat stress.