The genus Zoysia consists of 16 species that are naturally distributed on sea coasts and grasslands around the Pacific. Of these, Zoysia japonica, Zoysia matrella, and Zoysia tenuifolia are grown extensively as turfgrasses, and Z. japonica is also used as forage grass in Japan and other countries in East Asia. To develop simple sequence repeat (SSR) markers for zoysiagrass (Zoysia spp.), we used four SSR-enriched genomic libraries to isolate 1,163 unique SSR clones. All four libraries contained a high percentage of perfect clones, ranging from 67.1 to 96.0%, and compound clones occurred with higher frequencies in libraries A (28.6%) and D (11.6%). From these clones, we developed 1,044 SSR markers when we tested all 1,163 SSR primer pairs. Using all 1,044 SSR markers, we tested one screening panel consisting of eight Zoysia clones for testing PCR amplifications, from which five unrelated clones, among the eight, were used for polymorphism assessment, and found that the polymorphic information content ranged from 0 (monomorphic loci) to 0.88. Of the 1,044 SSR markers, 170 were segregated in our mapping population and we mapped 161 on existing amplified fragment length polymorphism-based linkage groups, using this mapping population. These SSR markers will provide an ideal marker system to assist with gene targeting, quantitative trait locus mapping, variety or species identification, and marker-assisted selection in Zoysia species.