A complementary DNA(cDNA) library was constructed from half-smooth tongue sole spleen. A long-read expressed sequence tag(EST) database was generated,containing 3100 cDNA clones,of which 220 clones were fully sequenced. A total of 1060 non-redundant simple sequence repeats(SSRs) were obtained from the cDNA library. An average of 5 kb sequence generates 1 SSR in the half-smooth tongue sole spleen cDNA library. The proportion of the SSR unit size was different in the cDNA library. The monomeric repeats(51.4%) are the most abundant class of SSR in the dataset. The dimeric,trimeric,tetrameric and hexameric re-peats are represented in decreasing proportions of 27.2%,16.0%,2.8% and 1.9%,respectively. The frequency of pentameric repeats was observed the least(only 0.7%) . Most of the monomeric and dimeric repeats are distributed in 3’ and 5’ un-translation region. If translation regions are considered merely,trimeric repeats are the highest,accounting for 57% of the total microsatellites. A long DNA readout sequence tag (EST) database was generated, containing 3100 cDNA clones, of which 220 clones were fully sequenced. A total of 1060 non- The average of 5 kb sequence generates 1 SSR in the half-smooth tongue sole spleen cDNA library. The proportion of the SSR unit size was different in the cDNA library. The monomeric The dimeric, trimeric, tetrameric and hexameric re-peats are decreasing in proportions of 27.2%, 16.0%, 2.8% and 1.9%, respectively. The frequency of repeats (51.4%) are the most abundant class of SSR in the dataset. Most of the monomeric and dimeric repeats are distributed in 3 ’and 5’ un-translation region. If the translation regions are considered merely, the trimeric repeats are the highest, accounting for 57% of the total microsatellites.