The olive flounder (Paralichthys olivaceus) is one of the most widely cultured flatfish in Korea and Japan. During development, in a process known as metamorphosis, this fish reorients itself to lie on one side, the body flattens, and the eye migrates to the other side of the body. However, few studies have focused on molecule regulation mechanism of eye development in olive flounder. To reveal the molecular mechanism of eye development, we performed the studies on identification of genes expressed in the eye of olive flounder using EST and RT-PCR strategy. A total of 270 ESTs were sequenced, and 178 (65.9%) clones were identified as known genes and 92 (34.1%) as unknown genes. Among the 178 EST clones, 29 (16.3%) clones were representing 9 unique genes identified as homologous to the previously reported olive flounder ESTs, 131 (73.6%) clones representing 107 unique genes were identified as orthologs of known genes from other organisms. We also identified a kind of eye development associated proteins, indicating EST as a powerful method for identifying eye development-related genes of fish as well as identifying novel genes. Further functional studies on these genes will provide more information on molecule regulation mechanism of eye development in olive flounder.