Expressed sequence tags (ETSs) are the sources of microsatellite development. In this study, we isolated and characterized microsatellite markers for Odontobutis potamophila by using Illumina RNA-sequencing. We sequenced a large number of ESTs and screened 200 potential microsatellites. Consequently, a total of 56 novel polymorphic microsatellite repeat markers were identified in thirty-two individuals from a wild population area (Jiande, Zhejiang Province, China). The number of alleles per locus varied from two to eight, the observed heterozygosity (HO) ranged from 0.03571 to 0.9375, and the expected heterozygosity (HE) ranged from 0.14326 to 0.81549. The average number of alleles, HO, and HE were 5.0, 0.4467, and 0.5518, respectively. By the calculation, the range of polymorphism information content (PIC) was 0.1177-0.8492. Most of the loci showed moderate or high polymorphism. These newly developed EST-simple sequence repeat (EST-SSR) markers would serve as an efficient tool for analyzing population connectivity and provide sufficient information for genetic diversity research, parentage, and molecular breeding of O. potamophila and other fishes with similar genetic relationship.