Twenty-four microsatellite markers were isolated from the genomic DNA of Amomum tsaoko Crevost et Lemaire, an important economic plant in China, using the method fast isolation by AFLP of sequences containing repeats (FIASCO). Polymorphism within each locus was assessed in 60 individuals from three populations in Yunnan Province, China, and nine of them were polymorphic. The number of alleles per polymorphic locus was 2, and the expected and observed heterozygosities ranged from 0.224 to 0.513, and from 0.050 to 0.600, respectively. Among nine microsatellite markers with polymorphism, five showed significant deviation from Hardy-Weinberg equilibrium (P < 0.01), probably due to anthropic selection and short-cloning history in cultivation. No significant linkage disequilibrium was detected between loci in our analysis. These polymorphic microsatellite markers will facilitate further studies of gene flow, population structure, identification of cultivated variety, and evaluation of germplasm resources.