Heteroplexis microcephala Y. L. Chen. is an endemic and endangered species found only in karst limestone regions in the Yangshuo County of the Guangxi Zhuang Autonomous Region in China: it is a habitat representative of species in the Heteroplexis genus. To provide basic genetic information for its conservation, in this study we evaluated the genetic variation and differentiation among six wild populations of H. microcephala by random amplified polymorphic DNA markers (RAPD). The leaves of 141 individuals were sampled. Based on 12 primers, 113 DNA fragments were generated. Genetic diversity was low at the population level (Nei’s gene diversity (h)=0.0579; Shannon information index (I)=0.0924; percentage of polymorphic bands (PPB)=23.30%), but relatively high at the species level (h=0.1701; I=0.2551; PPB=46.34%). The coefficient of genetic differentiation based on Nei’s genetic diversity analysis (0.6661) was high, indicating that there was significant genetic differentiation among populations, which was confirmed by AMOVA analysis exhibiting population differentiation among populations of 68.77%. Low gene flow among populations (0.2507) may result from several factors, such as a harsh pollination environment, population isolation and low seed dispersal distance. Limited gene flow and self-compatibility are the primary reasons for the high genetic differentiation observed for this species. We propose the collection of seeds from more populations with fewer individuals and core populations for ex situ conservation and suggest methods to increase seed germination rates.