To clone.the differential expressional genes between the pre-metamorphosis （17 day post hatching, DPH） and prometamorphosis stage （23 DPH）, we used suppression subtracfive hybridization （SSH） to construct a cDNA library of prometamorphosis flounder Paralichthys olivaceus, when the RNA from pre-metamorphosis larvae was used as the driver. The lengths of inserted cDNA fragments in the library ranged from 215 to 774 bp, and the average length was about 558bp, which is much close to the cut frequency of restrict endonuclease Rsa Ⅰ. The percentage of positive clones in the SSH library was 81.8%. Total 45 clones Were sequenced randomly. Using relative quantafive RT-PCR to check the gene expressional level, we thought that the positive clones should express＇at higher level in pro-metamorphosis flounder than that in pre-metamorphosis larvae, otherwise, they should be regarded as false positive clones. Total22 genes represent positive clones including 13 homologue genes with unknown proteins, of which most have high similar sequences with other organisms, and 9 homologue genes with known proteins like prolactin receptorlike, COL1A1,M3-muscarinic receptor, Sfrs3, tropomyosin, ribosomal protein L27, ribosomal protein S12, GAPDH, COL1A3. Among all the 22 positive genes, the frequency of COL1A1 clone was highest as 22.2%, the prolactin receptor-like was next to the highest as 8.9%, most genes distributed with lowest frequency 2.2% in SSH cDNA library. The results of RT-PCR showed that the 11 representative genes all expressed in the heads of pre-metamorphosis and pro-metamorphosis flounder larvae.