In chicken, the bipotential embryonic gonad differen-tiates into either a pair of testes or an ovary, but few genes that underlying the gonadal sex differentiation have been identified and the sex-determination gene is still unknown. To identify more genes involved in chicken sex differentiation, we employed suppression subtractive hybridization to isolate differentially expressed genes between sexes from chicken gonads during a period of E3.5-E6. A total of 152 cDNA clones corresponding to 88 genes (41 from F-M library and 47 from M-F library) were screened using dot-blot analysis. These genes are located mainly on the macro-chromosomes (1-5) with five in the sex chromosomes (one in W and four in Z), encoding four dominating molecular categories belonging to enzyme, DNA associ-ation, RNA association, and structural protein.Comparing the obtained cDNA sequences with those in chicken EST database, it showed that cDNAs of 32genes from F-M library and 16 from M-F library have homologs in two reported embryonic gonad cDNA libraries. Quantitative real-time PCR analysis of eight genes involved in epigenetic and transcription regu-lation showed significantly different expression between sexes of CDK2API, SMARCEI, SAPIS, SUDS3, and PQBPI appeared at the early stage in gonad develop-ment (E4). Based on the functional comparison of sexual differentially expressed genes, the roles of some putatively important genes including ATP5AIW,CDK2AP1, mitochondrial transcripts, etc. have been analyzed. In conclusion, characterization of isolated genes would provide valuable clues to identify potential candidates involved in genetic mechanisms of chicken sex differentiation and gonad development.