To identify known genes or expressed sequence tags (ESTs) which are expressed specifically or preferentially in the chicken hypothalamus and pituitary gland related to highly reproductive performance, two reciprocal cDNA libraries were constructed using a subtractive hybridization strategy. Two different strains, L2 (dam line; n=12) and B (sire line; n=12) of Taiwan Country Chickens (TCCs), which were originated from one single strain and further subjected to 40-wk egg production and comb size, body weight, respectively since 1982, were used in our study. A total of 324 and 370 clones were identified from L2-subtract-B and B-subtract-L2 hypothalamus/pituitary cDNA libraries. 311 and 360 single inserted sequences from each cDNA library, 53 and 23 non-redundant candidate genes were identified. Quantitative reverse-transcription (RT)-PCR were used to validate the association of mRNA expression profiles of the identified candidate genes and high-egg yield trait in another 118 hypothalamuses and pituitary glands that were dissected from seven different chicken stocks, including B-, L2-, Black-, Red-feather TCCs, commercial Single-Comb White Leghorn (WL) layer at National Chung-Hsing University (NCHU) and Red-feather TCCs grouped into high eggs (Red-high) & low eggs (Red-low) to 40 wks of age at National Chiayi University (NCYU). Among identified genes including known genes and novel genes, involving 33 screened genes, Inhibitor-1 of protein phosphatase type 2A (ANP32A), 3-hydroxybutyrate dehydrogenase (BDH), Contactin (CNTN1), Deiodinase iodothyronine type II (DIO2), Inhibitor of growth family, member 3 (ING3), Lysosomal-associated transmembrane protein 4 beta (LAPTM4B), Neural cell adhesion molecule 1 (NCAM1), DJ-1 protein (PARK7), Prostaglandin D2 synthase (PGDS), Prolactin (PRL), Protocadherin 1 (PCDH1), Pleiomorphic adenoma gene 1 (PLAG1), GTP-binding protein SAR1a (SAR1A), Secretogranin II (SCG2), Stathmin 2 (STMN2), T-box protein 2 (TBX2) were up-regulated in B-subtract-L2 cDNA library. Among above-mentioned 16 identified genes, there were 9 genes related to high-egg yield in chickens., including BDH, NCAM1, PCDH1, PGDS, PLAG1, PRL, SAR1A, SCG2, STMN2.