Abstract: The yellow perch, Perca flavescens, is an economically significant freshwater fish species in the Midwest of the United States. TypeⅠ markers are useful for comparative mapping and other genetic analyses, but limited quantities in yellow perch. In the present study, EST containing microsatellite sequences were identified and characterized for yellow perch by data mining from updated public EST databases. Out of 21968 EST sequences of yellow perch, about 14.4% of ESTs contain repeat motifs of various types and length. CA repeat was the most abundant dinucleotides. Out of the 62 EST-SSRs for which PCR primers were designed, 15 loci showed polymorphic in a yellow perch population with the alleles per locus ranging from 4 to 19 (average 9). The observed (H_O) and expected (H_E) heterozygosity of these EST-SSRs were 0.103—0.929 and 0.116—0.934, respectively. Four EST-SSR loci significantly deviated from the Hardy-Weinberg equilibrium (HWE) expectation, and the remaining 11 loci were in HWE. These new EST-SSR markers should provide sufficient polymorphism for population genetic studies and genomic mapping of yellow perch.