S-100 proteins are calcium-activated signaling proteins that interact with other proteins to modulate biological functions such as cell migration, growth, proliferation, differentiation, apoptosis, contraction, and the inflammatory response. Although S-100 proteins are expressed in neuronal and non-neuronal tissues, their localization in the uropygial gland was not known. This study concerns the immunohistochemical detection of localization of S-100α, S-100β, and wbS-100 in the chicken uropygial gland during development from days 1–150 days post-hatching. In 1-day-old chicks, the nuclei and cytoplasm of cells in the luminal epithelium and the tubules of the gland stained positively for S-100α, S-100β, and wbS-100. Seven days after hatching, immunoreactivity for S-100α, S-100β, and wbS-100 was detected in the nucleus and cytoplasm of cells in the germinative and intermediate layers of the central zone, but was found only in the germinative layer of the peripheral zone. In addition, in cells of the degenerative and secretory layers of both the central and peripheral zones, S-100α, S-100β, and wbS-100 were present in the plasma membrane. In uropygial glands studied from days 7–150 post-hatching, the number of immunopositive cells in the central zone increased with the advance of age and glandular growth. The results indicated that the chicken uropygial gland contains both αβ and ββ dimers. Although the biological significance of the expression of wbS-100 and its subunits in the uropygial gland remains unknown, these notable calcium-binding proteins may be associated with the processes of gland-cell differentiation and apoptosis, and the antimicrobial properties of preen wax or lipids.