Biological bases for sexual differences in the brain exist in a wide range of vertebrate species, including chickens. Recently, the dogma of hormonal dependence for the sexual differentiation of the brain has been challenged. We examined whether sexually dimorphic gene expression in the brain precedes gonadal differentiation. Using the Affymetrix GeneChip Chicken Genome Array, we identified 42 female- and 167 male-enhanced genes that were differentially expressed in sex-specific brains from stage 29 chicken embryos. To confirm the efficacy of the microarray, and to investigate the stage-specific expression patterns of the identified genes, we used quantitative real-time PCR analysis. Our real-time PCR results for the differentially expressed genes agreed well with our microarray results. Thus, we postulate that these genes have potential roles in the sexual differentiation of neural function and development in chickens.