Abstract:Genetic variability in 30 populations of three endemic Castanea (Tourn.) L. species (C. moUissima BI., C. seguinii Dode. and C. henryi (Skan) Rehd. et Wils. ) in China was investigated using isoelectric focusing in thin-layer polyacrylamide slab gels at 20 loci coding for 12 enzyme systems. C. mollissima was found to possess a significant higher value of genetic variability than that of the other two species. The percentage of polymorphic loci (P) and the expected heterozygosity (He) were 90.0% and 0. 311 at the species level, respectively; while P = 84.7 %, He = 0.295 at the population level. Comparison of the population genetic variability of C. mollissima in three regions of China, the populations from the Yangtze River valley showed a markedly higher mean expected gene heterozygosity. The results provided additional evidence that the Yangtze River valley, particularly in Shennongjia area, was the center of the genetic diversity of C. moUissima. Genetic relationships among populations and species were assessed by Nei genetic identity (I) and standard genetic distance (D), suggesting that C. mollissima and C. seguinii have a closer relationship, and genetic distances were correlated with geographical distances among populations. Genetic differentiation between populations in C. mollissima, C. seguinii and C. henryi was 7.5%, 10.9% and 22.1%, respectively, and the gene flow rate (Nm) was 3.20, 2.05 and 0.88 respectively. The study provides useful imfonnation for understanding of the origin and evolutionary events in genus Castanea and planning an effective conservation strategy.