Preserving genetic diversity and evolution potentials of the population is a key to successfully conserve an endangered species. Nyssa yunnanensis, is a critically endangered and range-restricted tree species under national Grade I protection in China, which has been listed in national grade I protection and projected in the Conservation of Species with Extremely Small Populations. To explore and draw up the pertinent conservation strategies, we examined the genetic diversity of 64 N. yunnanensis individuals using inter-simple sequence repeat (ISSR) markers generated by twelve ISSR primers. The results showed that the genetic diversity of natural population of Nyssa yunnanensis(PPB) ranged from 50.00% to 87.50% with a mean of 74.65%±0.01; Alleles observed value (Na) was 1.7532; Eeffective alleles observed value (Ne) was 1.5804; Nei's genetic diversity index (He) was 0.3206, Shannon’s index (I) of phenotypic diversity at the species level was 0.3630, Polymorphism information content (PIC) ranged from 0.15 to 0.44. with a mean of 0.27±0.01. Moreover, Shannon’s index (I) of phenotypic diversity raised as the number of N. yunnanensis population size increased. But Shannon’s index(I )I would had not been changed until the number reached 24. Based on results of genetic diversity analysis, we clarified the endangered mechanism of N. yunnanensis on the viewpoints of both geological history and contemporary human disturbances. Conservation measures were suggested to maintain and increase genetic diversity, evolution potentials, and environment adaptability of N. yunnanensis, so as to reach the goal of successfully conserving conserve this critically endangered species.