SNPs widely distribute throughout genomes from non-coding regions to coding regions, constituting the most abundant molecular markers used in animal and plant genetic breeding. With the rapidly growing genome sequencing projects, a large amount of genomic and EST sequences has become available to the public. Many SNPs are identified by comparing genome sequences or ESTs obtained from genetically diverse lines or individuals in plants. However the SNP assay always relies on expensive equipments or reagent, which has limited the application of SNPs in genetics and breeding especially in plants. The CAPS marker, also known as PCR-RFLP marker, is the technique combining PCR and restriction enzymes digestion to detect the restriction fragment length polymorphisms. With the development of high-throughput sequencing technology, more and more SNPs are identified, among them many mutations have altered the restriction enzymes recognition sites. This provides an opportunity for high-through development of CAPS markers. With soybean genome sequences becoming available, high-throughput SNP marker development will significantly improve genetic mapping, map based cloning in soybean. To discover new SNPs in soybean, we aligned the ESTs with whole genome sequences in different soybean varieties and identified 537 EST-SNPs. The function of genes targeted by these EST-SNPs was analysed, the results showed that these genes participated in subcellular localization, protein binding or catalyzing, metabolic process and cell rescue, defense and disease resistance, etc. Most of these functions are involving in various physiological and biochemical processes influencing important agronomic traits. To develop easy assay method for these EST-SNPs, we identified the EST-SNPs which alter the restrict enzyme recognition sites by software EMBOSS, and 48 pair primers were designed to detect these EST-SNPs. forty-four pair primers amplified single bands (400–800 bp) from genomic DNA of Suinong 14 widely planted in the Northeast China. To verify the SNP polymorphisms, we used these primer pairs for PCR amplification from genomic DNA of Suinong 14, Hefeng 25, Acher, Evans, Peking, PI209332, Guxin wild soybean, Kefeng 1, Nannong 1138-2 and pool DNA of the nine soybean varieties. The PCR amplicons were sequenced, the traces of the 36 discordant ones were detected as candidate SNPs, which were then validated by re-sequencing the individuals. SNPs were identified using restriction enzymess of 26 pair primers with unequivocal restriction pattern were identified as CAPS markers. The SNPs discovery and CAPS markers conversion system developed in this study is fast, low cost and effecient, and holds great promise for molecular assisted breeding of soybean. , and the product