Microsatellite-enhanced genomic library of Cymbidium sinense was constructed using repeat-enrichment method with biotin-labeled oligos and streptavidin magnetic beads. Twenty-five microsatellite loci were isolated from a microsatellite-enhanced genomic library of Cymbidium sinense. Nine of these loci displayed genetic diversity that ranged from one to 15 alleles per locus, and the observed heterozygosity of six polymorphic loci ranged from 0.18 to 0.90 with an average of 0.47 (0.29 sd) in a sample of 30 individuals. Eight of these loci were successfully amplified in at least one of the following species: Paphiopedilum hissutissimum, P. wardii, P. armeniacum, or P. micranthum. The study provides a base for molecular breeding and assessment of germplasm resources of the Cymbidium sinense and the results suggest that the microsatellite loci developed from C. sinense may have a broad applicability within the Orchidaceae family.