Zoysiagrass (Zoysia sp.) is extensively used in turf establishment and livestock herbage due to its many outstanding characters. Native Zoysia sp. are widely distributed in China. Inter-simple sequence repeat (ISSR) markers were used to investigate the genetic diversity and genetic relationships of 81 Chinese wild zoysiagrass accessions and three commercial cultivars. The results show that 33 ISSR primers produced 388 clear bands, among which 375 were polymorphic. The genetic similarity coefficients (GSCs) among 84 zoysiagrass accessions or cultivars ranged from 0.644 to 0.866 with an average of 0.751. The GSCs within species were significantly higher than that among species. Cluster analysis using an unweighted pair group method with arithmetic mean (UPGMA) method showed that the 84 zoysiagrass accessions could be classified into 10 major groups. Accessions from similar geographic regions were generally clustered together, which indicated a correlation between molecular groupings and the geographical origin. The investigation demonstrated the genetic diversity of different germplasm, and that ISSR markers are an effective tool for the study of genetic variation in zoysiagrass.