We developed novel and polymorphic microsatellite primers for Spathoglottis plicata, a tropical and subtropical terrestrial orchid, to investigate the genetic patterns and population structure among wild populations, and also to identify the varieties and hybrids of S. plicata in horticultural industry. The 12 novel microsatellites from S. plicata were developed by using polymerase chain reaction (PCR)-based isolation of microsatellite arrays. These markers that were successfully PCR amplified exhibited polymorphisms in S. plicata. The number of alleles, observed heterozygosity, expected heterozygosity and polymorphism information content values across loci ranged from 2.000 to 8.000, 0.000 to 0.756, 0.208 to 0.813 and 0.405 to 0.805 in total populations, respectively. The newly developed microsatellite markers exhibited variation in S. plicata. These markers can be used as a tool to further investigate the genetic diversity, conservation genetics and variety/hybrid identification of S. plicata.