Premise of the study:Fokienia hodginsii (Cupressaceae) is a Tertiary relict evergreen conifer of the monotypic genus Fokienia. Polymorphic microsatellite markers were developed to investigate its genetic diversity and population structure.Methods and Results:RNA transcripts of F. hodginsii were sequenced and de novo assembled into 85,818 unigenes, and 1892 simple sequence repeat (SSR) markers were detected from the unigenes. A total of 273 expressed sequence tag–SSR primer pairs were designed and tested, and 129 successfully amplified. Eleven displayed clear polymorphisms in F. hodginsii. Amplification of these polymorphic primers across three populations of F. hodginsii showed the number of alleles per locus ranged from two to seven, and the expected heterozygosity per locus varied from 0.067 to 0.847. All 11 polymorphic primers amplified in Thuja occidentalis, while 10 amplified in T. standishii, Platycladus orientalis, and Chamaecyparis obtusa.Conclusions:These microsatellite markers will be useful in exploring genetic diversity of F. hodginsii and other conifer trees.