Phoebe chekiangensis is a uniquely precious timber and ornamental tree species of Southern China that enjoys nationwide protection as an endangered species. In the present study, we identified a total of 22,985 SSR loci based on transcriptome sequencing data, and 41 polymorphic markers were chosen to analyze the genetic diversity of seven natural populations including 190 individuals. Results showed that a moderate level of genetic diversity was in this species. A UPGMA dendrogram revealed that the seven populations clustered into four categories, and the 190 individuals could be divided into five groups with the aid of STRUCTURE 2.3.1; the clustering tendency revealed using two distinct algorithms was similar. The genetic variation was mainly within populations, and there was frequent gene flow among populations. On Mantel testing, genetic distance was positively and significantly correlated with geographic distance. In terms of conservation, the WY, YS, SY, and HZ populations, which exhibited the highest genetic diversity, should be the prime targets of both in situ and ex situ conservation efforts. Populations TS, YJ, and LC, with fewer individuals and lower genetic diversity, are susceptible to anthropogenic interference and should be carefully protected in situ . Also, ex situ conservation and propagation are required.