Rice leaffolder, Cnaphalocrocis medinalis (Guenée), is a destructive and widespread pest on rice. In this study, 20 microsatellite markers were isolated and characterized from C. medinalis partial genomic libraries using the method of fast isolation by AFLP of sequence containing repeats. Of these markers, 18 markers displayed polymorphisms. Polymorphisms were evaluated in 48 individuals from two natural populations. The number of alleles per locus ranged from 2 to 15, and the expected and observed heterozygosities ranged from 0.324 to 0.934 and from 0.304 to 0.917, respectively. Cross-species amplification was also performed to test the transferability of the 20 microsatellite markers and a moderate level of cross amplication was observed across the three species of Pyralididae (26.67 %). These microsatellite loci would facilitate the future study on population genetics and molecular genetics of rice leaffolder and would also be useful for study in Chilo suppressalis, Scirpophaga incertulas and Pyrausta nubilalis.