Abstract: In order to study the germplasm genetic information of the late ripe varieties of Chinese chestnut in Luotian Hubei, in this paper, the classification and genetic relationship of 8 series of Chrysanthemum cultivars were investigated using cluster analysis of ISSR molecular markers. For the reaction system of ISSR-PCR and primer selecting, we selected the number of bands and early high-definition reaction system to screen 21 primers, eliminate primers of poor amplification and difficultly identified bands, and ultimately determine the 7 primers used for late ripe varieties of chestnut identification. For ISSR cluster analysis, the selected ISSR primers amplified 140 clear and reproducible ISSR bands, and 106 bands of them were polymorphic with a polymorphism rates up to 75.7%. With NTSYS 2.10e software, 8 Chinese chestnut cultivars can be divided into two categories by UPGMA cluster analysis at the dice similarity coefficient of 0.45. The analysis result showed that ‘Yangmaoli’ and ‘Guihuaxiang No.2’ had genetic differences with other late ripe varieties.