Abstract: In order to explore the genetic diversity and genetic relationship , 25 samples of wild Vulpia myuros were collected from different areas of Zhejiang province, and 1 sample was bought in the market. Intersimple sequence repeat (ISSR) was used to analyze their genetic diversity. The results demonstrated that from 100 pairs of primers, 15 pairs were selected with stable reaction system, clear amplification bands and more polymorphism. A total of 87 bands were amplified from 26 samples with the above selected 15 pairs of primers, among which 83 bands were effective, accounting for 95.40% of the total number of polymorphic bands. The genetic distance of the tested 26 samples were 0-0.707, the closest genetic distance was 0, indicating close in genetic relationship. Cluster analysis was carried out according to the genetic similarity coefficient. At 0.63, 26 germplasm of V.myuros could be clustered into two groups. One group consisted of 25 samples and another one had only 1 sample collected from Lishui. At 0.78, the 25 samples from the group could be subdivided into two categories: sample from Japan, and the other 24 samples.