Abstract: Abstrat: In order to analyze the genetic diversity of Aesculus chinensis var. Wilsonii germplasm resources in the Shennongjia Forest District this study utilizes Aesculus chinensis var. Wilsonii samples collected from Shennongjia (SB in Songbai Town, YR in Yangri Town, XH in Xinhua Town, SL in Songluo Township, MY in Muyu Town, HP in Hongping Town, XGP in Xiaguping Tujia Township, DJH in Dajiuhu Town) and Xiangyang City (XY) as materials. ISSR molecular markers were used to study the genetic diversity of 153 samples from these 9 Aesculus chinensis var. Wilsonii populations, and provide reference for the scientific protection and utilization of Aesculus chinensis var. Wilsonii, The results of the study are as follows: A total of 205 bands were obtained by ISSR marker analysis, of which 95.95% were polymorphic bands. The coefficient of genetic differentiation Gst was 0.2729, indicating that the genetic variation within the population was 72.21% and the genetic variation between the populations was 27.29%. Gene flow Nm = 1.3323, indicating a certain degree of gene exchange between populations. The genetic diversity within the SB and YR populations was the most abundant, followed by XY, while the genetic diversity within the XGP population was the lowest. SB and YR are the most closely related, and XGP and HP are the most distantly related. When the Euclidean distance was 3.58, the 9 groups were divided into 5 categories, and the first group was SB, YR, SL and XH. The second category includes MY and XGP; XY, HP, and DJH are in a separate category.