Purpose: We aimed to dissect response of Perennial_ryegrass to drought, heat and cold stresses and identify stress responsive genes in Perennial_ryegrass. Methods: The sequencing libraries was constructed using NEBNext® UltraTM RNA Library Prep Kit for Illumina®, sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated. Clean data (clean reads) were obtained after removing reads containing poly-N or adapter, and low quality reads from raw data. Transcriptome assembly was accomplished based on the left.fq.gz and right.fq.gz using Trinity. Gene function was annotated based on the following databases: NR (NCBI non-redundant protein sequences), Pfam (Protein family), KOG/COG/eggNOG (Clusters of Orthologous Groups of proteins), Swiss-Prot (A manually annotated and reviewed protein sequence database), KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology). Differential expression analysis of two groups was performed using the DESeq R package (1.10.1). Genes with an adjusted P-value<0.05 and fold change>2 found by DESeq were assigned as differentially expressed. Results:In total, four samples with two biological replicates per genotype/treatment combination were used for RNA sequencing analysis. At least 5 G clean bases were generated for each sample.After sequencing quality control, a total 137822219 clean reads and 41.05 G clean bases were generated. The Q30 base percentage of each sample was higher than 91.0% (Table S1). A total of 32840 unigenes were obtained after transcriptome assembly. Comparative analysis identified genes modulated by drought, heat and cold stresses Overall design: For cold and heat treatments, 14-day-old seedlings were treated at 4 and 42 oC for 12 hours, respectively. For drought treatment, 7-day-old seedlings were withheld water for 7 days. Stress-treated and control seedlings were collected for RNA isolation with 30 seedlings for each treatment or control replicate.