Abstract: Nine SSR markers developed by a modified FIASCO (Fast Isolation by AFLP Sequences COntaining repeats) method were used to analyze the genetic diversity and differentiation of three Osmunda japonica Thunb. populations,including two wild populations from Lushan and Enshi and one cultured population from Enshi. A total of 47 alleles were detected in the three populations, with a mean N_A(number of alleles) of 5.222. The observed heterozygosity and expected heterozygosity were 0.000-0.944 and 0.577-0.834, respectively. The Shannon index ranged from 0.962 to 1.860. Results suggested that the microsatellite loci were highly polymorphic. For each population, the average expected heterozygosity was higher than that of the observed heterozygosity, and the population inbreeding polymorphism coefficient was positive, indicating the existence of non-random mating. For the three populations, the wild population from Enshi harbored the most abundant genetic diversity, while the cultured population had the lowest. Low levels of genetic differentiation were found between the two wild populations (F_ST＝0.092), which was supported by AMOVA analysis.