Expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers could enrich the current resource of molecular markers. In this study, microsatellite markers were developed for Dendrobium nobile Lindl. by mining the ESTs. Twenty-eight EST-SSRs amplified 2 to 6 nucleotide repeats with a mean number of 2.82 alleles per locus. The observed and expected heterozygosities per locus ranged from 0.158 to 0.579 and 0.422 to 0.752, respectively. These novel EST-SSRs enriched the current resource of molecular markers for the Dendrobium genus and would facilitate further applications in germplasm appraisal, evolution and genetic diversity studies, genetic mapping, and plant breeding of D. nobile and other congeneric species.