By using suppression subtractive hybridization (SSH), subtracted cDNA libraries were constructed from rice field eel () gonad at IV ovary and ovotestis stage. Two-directional (forward and backward) SSH was performed under cDNAs of rice field eel gonad at IV ovary and ovotestis stage. The cDNA fragments were inserted into plasmid vectors, and then transformed into DH5α. Finally, forward and backward subtracted cDNA libraries (461 and 674 clones, respectively) were obtained. By PCR detection, length of the subtractive cDNA fragments cloned into the vector ranged from 200 bp to 2 000 bp. One hundred positive clones were selected and sequenced randomly from the forward and the backward libraries, and ninety gene fragment sequences were obtained. EST analysis of these sequences was performed with Blastx and Blaxtn by comparing sequences with GenBank database. Two sequenced genes were further analyzed by semi-quantitative RT-PCR method. The results showed that the library can enrich differentially expressed genes in the two phases gonad.