In order to reveal the genetic diversity and relationships of Cymbidium ensifolium varieties and provide evidence for utilization and exploitation of germplasm resources of C. ensifolium, sequence-related amplified polymorphism (SRAP) was used to analyze the genetic relationship among the 47 varieties, which derived from Sichuan, Taiwan, and Guangdong. The software NTSYS was applied to analyze the results of SRAP-PCR. A total of 188 DNA bands was amplified using 12 specific and stable primers selected from 88 primers, 147 of which was polymorphic. The average number of polymorphic DNA bands amplified by each primer was 12. Clustering analysis revealed that these varieties were divided into four groups. Group I consisted of 3 varieties from Sichuan. In group II, 18 varieties came from Sichuan, 3 varieties from Guangdong Province, and the remaining 2 varieties from Taiwan. In 12 varieties of group III, eleven varieties came from Taiwan, and only one originated from Sichuan. In the last group, 9 varieties were included, 8 of which came from Sichuan and 1 variety originated from Taiwan. These result indicate study the genetic diversity and relationship of C. ensifolium effectively.