Zoysiagrass (Zoysia spp.) is a perennial warm-season grass (C4 photosynthetic system), which is widely distributed in Africa, Asia and America. Of these, five species, Zoysia japonica, Zoysia matrella, Zoysia tenuifolia, Zoysia sinica and Zoysia macrostachya, have been identified from southern Hokkaido to southwest islands in Japan. We collected 258 ecotypes throughout Japan and investigated 5 morphological traits, leaf width and length, plant height, plant length, and runner length and internode length, and also analyzed the genetic diversity among them based on microsatellites. We found these ecotypes could be classified into 3 species, Z. japonica, Z. matrella and Z. tenuifolia, except 50 ecotypes based on these traits. A total of 52 putative alleles were detected using 12 microsatellite primer pairs. The minimum and maximum scores of gene diversity for individual loci were 0.2287 and 0.9246, respectively. The all ecotypes were divided into 2 clusters based on STRUCTURE analysis. In the each species, Z. japonica, Z. matrella and Z. tenuifolia were assumed to be divided into 2 or 3 clusters, respectively. The unweighted squared genetic distance based on the microsatellite data for all ecotypes were subjected to a principal coordinate analysis. The first two principal coordinates explained more than 30% of the variation in the estimates of genetic similarity. Overall, three distinct groups were identified by the first two principal coordinates and each groups were mainly contained Z. japonica, Z. matrella and Z. tenuifolia, respectively.