Objective: To investigate the genetic diversity and structure of Dendrobium huoshanense, a (CT)n enriched microsatellite library was constructed using a magnetic beads enrichment procedure. Method: The 3''-biotinylated oligonucleotide probe was used to hybridize with the digested D. huoshanense genomic DNA fragments whose both ends were ligated with adaptors. The hybridized complex was then combined with the streptavidin-coated magnetic beads. The captured microsatellite fragments were eluted, collected and cloned into pMD19-T vector. The recombinant plasmids were transformed into Escherichia coli DH5α competent cells. The clones that yielded two or more bands contained microsatellite fractions. Positive clones were screened and sequenced. Thirty pairs of primers were designed and synthesized. Polymorphism at each locus was determined using 24 individuals from a natural population from Huoshan county town in Anhui province. Result: Twelve polymorphic microsatellite loci from the microsatellite-enriched genomic library were newly developed across 24 D. huoshanense individuals. In total, 65 alleles were identified, and the number of alleles per locus ranged from 2 to 8. The mean observed and expected heterozygosities were 0.500 and 0.638, respectively. Two loci significantly deviated from Hardy-Weinberg equilibrium (P<0.05), which could be due to the presence of null alleles. Furthermore, three of twelve loci showed significant linkage disequilibrium (P<0.05). Conclusion: These results suggest that the identified polymorphic microsatellite markers will be useful in population genetic studies of D. huoshanense.