First, 75,160 raw Sanger EST sequences from Italian ryegrass cultivar Waseaoba were assembled into 8,983 contigs and 21,189 singletons. And then 10G raw data of 100bp pair-end Hi-Seq 2000 genomic reads from the three samples were aligned to the assembled EST contigs and singletons. The results showed that about 6-10% of the reads could be aligned to the EST contigs. A total of 88,990 putative SNP sites between the three samples were detected during the alignment of the three genomic samples to the EST contigs/singletons. We have currently tested 106 primers pair designed from randomly selected SNP sites by using high resolution melting (HRM) analysis based on BIO-RAD CFX96 machine, and found 88 primers can be worked well. More numbers of SNPs will be tested and mapped on a BC1 population derived from a cross between L. temulentum and Italian ryegrass. The SNP markers developed in this study will be useful for genome mapping, quantitative trait loci analysis, and genetic diversity studies in Lolium species.