There are a variety of approaches to identify groups of genes that change in expression in response to a particular stimulus or environment. We here describe the application of suppression subtractive hybridization (SSH) for isolation and identification genes in the brain of common carp (Cyprinus carpio) under cold temperatures. The materials were prepared through cooling the hybrid F2 of purse red carp (cold-tolerant strains) and bighead carp (cold-sensitive species) to different regimes of temperatures. A subtracted cDNA library containing 2000 clones was constructed. About 60 positive clones were identified to express differentially by dot blotting in screening 480 clones. Sequencing 26 clones and aligning in GenBank/EMBL database using blastn searching engine, 15 genes showed higher similarities with 85-98%. These annotated genes contained (1) genes for transcription factors and gene products involved in signal transduction pathways such as zinc-finger protein, brevican; (2) genes involved in lipid metabolism such as Acyl-CoA synthetases, and (3) genes involved in the translational machinery such as cytochrome c oxidase, ependymin glycoprotein. In addition, real-time PCR was also conducted to validate these genes. To sum up, we believe this study will make an important contribution to elucidate the possible mechanisms on fish cold tolerance at a molecular level.