Abstract: Expressed sequence tags (ESTs) may serve as an effective source for identifying type Ⅰ microsatellitemarkers. In the present study, the database containing 3027 ESTs from head kidney of grass carp was screened for typeⅠmicrosatellite sequences using Tandem repeats finder v4.0 software. A total of 322 type Ⅰ microsatellites were identified,among which 151 were the type of dinucleotide repeat being the most abundant microsatellite and accounting for46.9% of the identified microsatellite markers, and 137 were the type of trinucleotide accounting for 42.5%, while hexa-,penta- and tetra-nucleotide repeats were rare. Among the dinucleotide repeats, AC/GT was the most abundant type withpercentage of 50.3%, followed by the AG/CT type with 40.4%, while AT and GC were rare. The ESTs were used todesign primers to amplify grass carp genomic DNA, and polymerase chain reaction products of the ESTs were analyzedto determine the length of polymorphic sequences, and then 27 microsatellites were selected for polymorphism analyses.10 of the 27 primer pairs resulted in PCR products of expected size, of which 4 loci showed polymorphism in a wildpopulation of grass carp, with 2 loci having high polymorphism. The allele number of all the loci was 23, and the meanPIC and heterozygosity values were 0.5236 and 0.5441, respectively. It is concluded that these type Ⅰ microsatellitemarkers can be used for further study in genetic mapping and quantitative trait loci (QTLs) identification in grass carp.