To clarify the appearance of and chronological changes in two different gonadotropic hormone (Gth) cells, we examined the dynamics of Gth cells in detail during gonadal differentiation and development in the d-rR strain of medaka (Oryzias latipes). Expression of the sex-determining gene Dmy was evident in gonadal somatic cells at 5days post-fertilization (dpf). Glycoprotein-α (Gpa)-positive cells first appeared in the pituitary at 4dpf, regardless of genetic sex, while follicle-stimulating hormone-β (Fshb)-positive cells was detected in XX and XY embryos at 5 and 6dpf, respectively. In contrast, luteinizing hormone-β (Lhb)-positive cells were observed in both sexes of medaka after 70days post-hatching (dph). The density of Fshb-positive cells in the pituitary was significantly and transiently higher in XX than in XY fry at 0dph, and thereafter no significant differences were detected before sexual maturation. In this study, temporal expression of Fshb was observed, indicating that Fsh cells become differentiated before hatching and that sexual dimorphism in Fsh cells occurs transiently after sex determination in medaka.