In order to strengthen the protection and utilization of Cymbidium resources in China， ISSR markers were deployed to conduct the genetic diversity and fingerprint construction in this study. A total of 67 bands were detected with 11 primers in 96 samples， and the average of polymorphic bands rate is 73.63%， the number of alleles （N_a） is 1.925， the number of effective alleles （N_e） is 1.450， the genetic diversity of Nei's is 0.277， and the Shannon index （I） is 0.427， the percentage of polymorphic loci （PPL） is 92.54%， the genetic diversity within populations （H_s） is 0.1934， the genetic differentiation coefficient （G_st） is 0.3009， the total genetic diversity （H_t） was 0.2767， and the average gene flow among populations（N_m） of Cymbidium is 1.1619， the fixation index of pairwise genetic differentiation among populations ranged from 0.002 to 0.527， with an average of 0.325. The systematic clustering results indicate that Cymbidium has high degree of genetic differentiation， which was divided into three groups by systematic clustering analysis， Cymbidium. goeringii and C. sinense were assigned to one category， while C. kanran， C. goeringii var. longibracteatum， C. faberi， C. tortisepalum and C. ensifolium were in the second category， the hybrids were in a unique category， which has a large genetic distance from the other two categories. Principal coordinate analysis indicated that C. tortisepalum and C. goeringii showed a distant genetic relationship. 6 pairs of primers were selected to construct fingerprint QR codes for 96 species. Collectively， this study provided an important basis for the breeding and variety identification of Cymbidium in the future.