Although chicken spermatogonial stem cells (SCs) are important in spermatogenesis and transgenic research, little is known about these cells. Recently, our group constructed an in vitro culture system to establish germline stem cells (GSCs). To examine the mechanism of chicken spermatogonial SC development, we constructed a proteome map of GSCs from 4-week-old chicken testes. Soluble extracts of the GSCs were fractionated by 2-dimensional gel electrophoresis (pH 4-7). Several protein spots, including those that displayed significantly high levels, were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Of the 82/250 GSC spots examined, 56 yielded mass spectra that matched avian proteins found in the on-line databases. All of the identified proteins were classified into functional groups. This type of proteome map is an important resource for research on spermatogenesis and transgenesis.