Cross-species amplification of 55 microsatellite loci developed in european chestnut (Castanea sativa Mill.) and japanese chestnut (C. crenata Sieb & Zucc.) was tested in three chestnut species from China [C. mollissima Blume, C. seguinii Dode, and C. henryi (Skan.) Rehder & Wilson]. Among all the tested loci, 47 (85.5%), 47 (85.5%), and 44 (80%) were successfully amplified in each of the three Chinese species, respectively. All microsatellite loci tested from C. crenata successfully amplified in the Chinese species, while only 80.5%, 80.5%, and 73.2% of the loci originating from C. sativa amplified in the three Chinese species. The level of polymorphism and mean number of alleles was 58.2% and 4.12 for C. mollissima, 60% and 4.64 for C. seguinii, and 60% and 4.76 for C. henryi, with mean observed heterozygosity ranging from 0.440 to 0.549 and mean expected heterozygosity ranging from 0.506 to 0.615. Transferability of Castanea Mill. microsatellites provides a powerful tool for chestnut breeding programs and conservation genetic studies of Castanea species.