Genetic diversity and genetic relationships of 16 natural populations of Castanea henryi. were assessed by ISSR. 379 loci were identified in 449 individuals, of which 378 loci were polymorphic, the polymorphic loci of percent (PPL) was 99.74%. The mean expected heterozygosity (He) and the Shannon information index (I) was 0.2950 and 0.4522, respectively. In population level, the PPL was 46.09%. The genetic diversity in different population was not consistent. Than those from other 15 regions, Xiangxi region had the higher level of diversity (PPL=53.30%，He=0.1861，I=0.2781）. A high level of genetic differentiation was detected among populations based on Nei`s （Gst =0.4466）and the analysis of molecular variance (AMOVA), the distribution of genetic differentiation within and among populations was 52.51 %and 47.49%, respectively. 16 populations were clustered 2 groups and 5 subgroups by the UPGMA analysis. Xiangxi region maybe the secondary distribution center and (or) modern center of genetic diversity of Castanea henryi, also is the source center. It is the most valuable gene pool，therefore, natural populations of Castanea henryi in this region need deserve prior conservation.