Annual bluegrass (Poa annua) typically exhibits early or more severe turf quality decline than creeping bentgrass (Agrostis stolonifera) during summer months when they are co-present on golf course putting greens. The mechanisms underlying the difference in heat tolerance between the two species are largely unknown. This study was conducted to compare physiological responses between P. annua and A. stolonifera and to identify metabolites and associated metabolic pathways underlying the differential heat tolerance. Plants were grown in controlled growth chambers at 22/17 degrees C (day/night, non-stress control) and 35/30 degrees C (heat stress) for 42 d. Visual turf quality, percent green canopy cover, and leaf electrolyte leakage were measured weekly. Metabolomic analysis was performed with leaf tissues collected at 42 d of heat stress. Relative to control plants, heat-stressed P. annua exhibited more severe declines in health across physiological parameters than A. stolonifera. Comparative metabolomic analysis of heat-stressed plants relative to the respective control identified metabolites that were upregulated uniquely or to a greater extent in A. stolonifera (sucrose, stachyose, raffinose, and glucose, glucuronic acid, and malonic acid) or P. annua (proline, tryptophan, lysine, phenylalanine, tyrosine, valine, isoleucine, and leucine) and those uniquely downregulated (malate, fumarate, pyruvate, aconitic acid, malonic acid, lactate, and glucose-1-phosphate and glucose-6-phosphate) in P. annua. Those distinct metabolites are mainly in photosynthesis, respiration, secondary metabolism, and stress protection. They were associated with the difference in heat tolerance for A. stolonifera vs P. annua and could be used as biomarkers or incorporated into biofertilizers to improve P. annua heat tolerance.