As an ancient and endemic plant in China, Chimonanthus praecox has been widely distributed in southern China in the past. The population number of C. praecox has declined significantly because of habitat fragmentation and human influence. Thirty-one EST-SSR markers were developed from the species’ EST sequences and eight polymorphic EST-SSR markers were chosen to analyze the genetic diversity and structure of ten natural populations. The results indicated that the genetic diversity levels were low among populations and genetic variations of populations were mainly emerged among groups. F-statistics revealed great genetic differentiation among populations (FST = 0.270), albeit the moderate gene flow existed (Nm = 0.676). Complex genetic differentiation and genetic structure of C. praecox were determined with UPGMA cluster tree and Bayesian clustering analysis. Five barriers were implied in the species’ geographical distributions using the Landscape Shape Interpolation and Monmonier's Maximum Difference Algorithm analysis. According to the genetic results and habitat-dependent characteristics of the species, in situ conservation strategy was suggested to all populations.