Abstract: To expand the application of molecular markers in the analysis and identification of oat germplasm resources, EST-SSR markers were developed and utilized using publicly available oat EST data. After splice and redundancy check, 11618 sequences were obtained, and 556 ESTs containing 595 microsatellites loci were selected and assembled from 25376 oat EST sequences. Firstly, 50 primers were designed based on 556 ESTs, and 40 primers were screened. The amplified products were tested and sequenced using four primer pairs on five oat germplasm resources. The results showed the polymorphisms of bands were caused by differences in SSR. Forty effective primer pairs were tested on the genetic diversity of 15 hexaploid oat germplasm resources, and the study of transferability was analyzed among oat species. Eighty-nine loci were successfully amplified in 15 hexaploid oat germplasm resources, with an average of 2.23 loci per primer pair. Based on genetic similarity coefficient (Dice=0.93), the UPGMA dendrogram grouped 15 hexaploid oat germplasm resources into three clusters. The 15 germplasm resources were clustered according to species, and were sub-clustered following geographic origin. The EST-SSR primers showed higher availability on the study of chromosome ploidy of 31 unknown oat germplasm resources. Results showed some diploids and tetraploids of oat germplasm resources were tested in the genetic analysis of 31 oat germplasm resources. This study proved that EST-SSRs are valuable for genetic analysis, linkage mapping and transferability study among oat species. The EST-SSRs also proved useful in discriminating different species and different genome ploidy in oat.