The pars tuberalis (PT) is a part of the anterior pituitary gland that is located as a thin cell layer surrounding the median eminence. The characteristics of PT, including cell shape and cell composition, differ from those of the pars distalis (PD), suggesting that PT has unique physiological functions and different morphogenesis compared to PD. In this study, we used chicken embryos and showed for the first time that most hormone-producing cells in PT at embryonic day (E) 20.0 were only glycoprotein α subunit (αGSU)-positive staining cells. Then, using serial frontal and sagittal sections, we examined the detailed distribution of the αGSU mRNA-expressing region, as a marker of PT in the chicken embryonic pituitary gland during the E3.0-20.0 period. This three-dimensional expression pattern analysis clarified that αGSU mRNA expression initially appeared only in the bilateral regions of the Rathke's recess (RR) at E3.5, and this region expanded and showed a ring-like structure on RR. Subsequently, this αGSU mRNA-expressing region gradually expanded upward and reached the diencephalon at E8.0. This region became thinner as it surrounded the base of the diencephalon from E12.0 to E20.0. In this study, we demonstrated the detailed morphological changes of the chicken PT primordium by detecting αGSU mRNA, and we also showed that PT is a unique region in the early developmental stage.