Bentgrass is one of the most important cool season grass in the world. High temperature is one of the important factors adversely affecting bentgrass growth in summer. To gain a better insight into the molecular mechanisms by which plants tolerate heat stress, we generated a suppression subtractive cDNA library to identify genes possibly involved in heat stress responses in a thermal rough bentgrass, Agrostis scabra. After subtraction, the cDNA fragments were cloned and two differential screening steps were used to screen the heat stress-induced library, resulting in 288 putative heat stress responsive cDNAs out of a pool of 1180 clones. All 288 cDNAs were sequenced and used to search for identification against the GenBank database. BLASTx and BLASTn analysis revealed an array of 125 non-redundant genes, of which 78 showed a high degree of sequence homology to known and unknown proteins or genes in the database. The genes were categorized into six functional groups, including signaling/ transcription, stress/defense, protein metabolism, carbon metabolism, other metabolism and unknown, indicating that complex alterations occur during heat stress in Agrostis scarbra. The largest group of genes with known function had homology to genes involved in stress/defence, followed by the group of genes related to protein metabolism. Of the identified clones, 21 (26.9 %) had homology to genes with unknown functions. Fourteen genes were selected for further Northern analysis to ascertain the data from the SSH library. Results showed that transcripts of all the genes were strongly increased by heat stress, with differently spatial expression patterns. Our study illustrates complex changes of gene regulation during heat stress and we conclude that these heat stress responsive genes are coordinately regulated under heat stress conditions, resulting in enhanced heat tolerance in Agrostis scabra.