Microhyla fissipes, from family Microhylidae suborder Neobatrachia, widely distributes from western Myanmar eastward through Indochina and northward into southern China including Hainan and Taiwan. But the availability of microsatellites especially from expressed sequences is currently very limited in this species and the genus Microhyla. A total of 17,339 potential microsatellites were identified in 15,385 unigenes which were generated by Illumina paired-end sequencing in M. fissipes. Within all microsatellites, AG/CT, AAG/CTT, and AAAG/CTTT are most prevalent motif in each repeat class. We randomly selected 61 unigenes with the microsatellite to design primers and do genetic analysis in the Sichuan basin population of M. fissipes. Of all, 35 primer pairs (57.38%) successfully amplificated in M. fissipes, of which 14 (40.00%) were polymorphism. The observed and expected heterozygosity in the test population ranged from 0.02 to 0.92 and from 0.02 to 0.62, respectively. High transferability rates were detected in M. butleri (37.14%), M. heymonsi (45.71%), M. pulchra (57.14%)and M. mixture (71.43%).These results indicate that the Illumina paired-end sequencing system is of great value for identifying massive numbers of genic microsatellites in M. fissipes with high-efficiency. Furthermore, the described polymorphic loci in this study should be useful for population genetic and conservation genetic studies in M. fissipes and other closely related species from this important genus Microhyla.