Perennial ryegrass (Lolium perenne L.) is a popular turfgrass species. To understand the molecular mechanisms of salinity tolerance, a suppression subtractive cDNA library was constructed for a salinity-tolerant ryegrass accession, with NaCl-treated (255mM) plants as the tester. Differentially expressed cDNA fragments were cloned and screened. BLAST search revealed that 268 clones exhibited significant homologies to known genes. These genes could be categorized into 11 different functional groups, including metabolism, energy transfer, detoxification, compatible solute, cellular transport, transcription, signal transduction, etc. The salinity-regulated expression of selected genes was confirmed by RT-PCR analysis. The results suggested that these putatively salinity up-regulated genes may play a vital role in the salinity tolerance of perennial ryegrass. They can be used as candidate genes for creating stress-tolerant grasses and for understanding molecular mechanisms of plant adaptation to salinity stress.