Ninty-one individuals of eight populations of Machilus thunbergii collected from Zhoushan archipelago were examined by inter-simple sequence repeat (ISSR)markers to determine the genetic structure. A total of 75 discernible loci were obtained using 9 primers, of which 61.3%were polymorphic (PPL=61.3%). As analyzed by POPOGENE, average values of preeintage of bolymorphic loci (PPL=52.3G) indicted that M. thunbergii had a reduction in genetic diversity compared with Taiwan populations. The value of differentiation (GST=0.311) affirmed that M. thunbergii had high genetic differentiation among populations. The effect of geographical isolation had great influences and significant correlation was found between genetic distance and geographic distance (r=0.655, P=96.1 0%). UPGMA indicated that Putuo Island population showed a close relationship with Zhujiajian Island populations, Damao Island population and Taohua Island populations clustered in a elade. In order to maintain genetic diversity and resource utilization of M. thunbergii, management strategies were proposed that natural population should be protected in situ from human dist’urbanee to facilitate its natural generation. Constructing germplasm resources and cultivating plants mutually collected from different populations are necessary to enhance the gene flow.