In chickens, although estrogen receptors (ER) are reported to be associated with the immunological processes, detailed information about the differences in ER expression in the tissues related to the development of lymphocytes is not fully known, especially during the developmental stage. To learn more about this immunological relationship, we used semi-quantitative polymerase chain reaction method to detect the ER expression levels in the thymus tissues of chicks during the developmental stage. Furthermore, ER-expressing cells were detected by immunohistochemistry. The results of this study show that the expression level of ER increased on embryonic day 16 and decreased on day 20. Furthermore, ER expression was significantly higher in male than in female chickens at day 16. The increased expression on day 16 and decreased level on day 20 were also reproduced in the incidence of immunoreactive cells, although there was a 1-day delay in the elevated incidence of the cells. This study revealed the changes in ER expression and the incidence of ER-positive cells in the thymus of chickens during the developmental stage.