Paeonia lactiflora is a valuable natural resource for horticulture and traditional Chinese medicine. To propose conservation strategy and future utility of the wild Paeonia lactiflora populations recently found around the Gyeongju National Park, genetic diversity analysis using microsatellite markers were performed. Three populations in and near the Gyeongju N.P. and one population from Jilin, China were analyzed for five microsatellite markers, producing 61 alleles with mean observed heterozygosity(Ho) of 0.452. FST value (0.11642) suggested moderate level of genetic differentiation among the populations, and hierarchical AMOVA suggested most of the genetic variation resides within/among the individuals rather than among-population. While AMOVA with FST suggested lack of genetic differentiation between the regional (Korean vs. Chinese) populations, AMOVA with RST, which incorporates the allele sizes, suggested considerable differentiation between them, but without significant statistical support. STRUCTURE analysis also suggested segregation of regional populations with presence of gene flow among the three Gyeongju N.P. populations. Considering small population size and scarcity of mature individuals, further protection and long-term monitoring are needed.