We have constructed a cDNA library using brain samples of olive flounder Paralichthys olivaceus. Here, we described the study on gene identification by screening 356 clones from the brain cDNA library of olive flounder. Here, we screened 356 clones from the library to identify genes. Of these, 176 (49.5%) were identified as orthologs of known genes from olive flounder and other organisms. Among the 176 EST clones, 33 (18.7%) represented 11 unique genes that are identical to expressed sequence tags (ESTs) reported for olive flounder, and 120 (68.2%) represented 102 unique genes known from other organisms. The percentage of unknown genes (50.5%) is higher than in other olive flounder cDNA libraries (Lee et al., 2003, 2006, 2007), reflecting the high complexity of brain tissue. Further studies of expression characterization and developmental behavior related to these genes should provide useful insight into the physiological functions of the brain in olive flounder.