Protein metabolism plays important roles in plant adaptation to heat stress. The study was designed to identify heat-responsive proteins in two C3 grass species contrasting in heat tolerance: heat-tolerant Agrostis scabra Wild and heat-sensitive Agrostis stolonifera L. Plants of both species were exposed to 20oC (optimal temperature control), 30oC (moderate heat stress) or 40°C (severe heat stress) in growth chambers. Roots were harvested at 2 and 10 d after each temperature treatment. Proteins were extracted from roots and separated by two-dimensional polyacrylamide gel electrophoresis. Seventy protein spots were differentially expressed in roots exposed to heat stress compared to those roots grown at 20oC in both species. Under both moderate and severe heat stress, significantly more proteins were down-regulated compared to up-regulated proteins. Heat-tolerant A. scabra roots had more up-regulated proteins than A. stolonifera roots. The sequences of 66 protein spots were analyzed using mass spectrometry. Sequencing analysis revealed increases in the accumulation of sucrose synthase, glutathione S-transferase, superoxide dismutase, and heat shock protein Sti (stress inducible protein) under heat stress, suggesting these proteins may contribute to root thermotolerance of heat-tolerant A. scabra. In addition, phosphoproteomic analysis showed that two isoforms of fructose-bisphosphate aldolase were highly phosphorylated by heat stress, and heat-tolerant A. scabra had greater phosphorylation than A. stolonifera, suggesting that the aldolase phosphorylation might involve in root tolerance to heat stress.