Cycas debaoensis is one of the critically endangered cycad species endemic to China. In this study, we described the development of six microsatellite markers from the genome of C. debaoensis using the protocol of fast isolation by AFLP of sequences containing repeats (FIASCO) and two microsatellite markers derived from the database of expressed sequence tags (dbEST). Polymorphism of each locus was assessed in 60 adult individuals of the cycad. The average allele number of the microsatellites was 2.6 per locus, ranging from two to five. The observed and expected heterozygosities varied from 0.0833 to 0.7333 and from 0.0805 to 0.7188, respectively. Despite its rarity, only one locus (Y177) deviated from Hardy–Weinberg equilibrium due to the excessive homozygosity. The marker transferability of the eight primer pairs was tested on other four congeneric species that also occur in China.