Over the past few decades, Medaka (Oryzias latipes) has become a model animal in teleost species research due both to its short reproductive cycle and efficient proliferate capacity. Unfortunately, however, systematic data of its sexual differentiation and development have yet to be obtained. In the present study, we observed Medaka gonad development from the earliest recognizable stages through differentiation to maturation under a light microscope, after paraffin sectioning and hematoxylin-eosin staining. The results showed that among juveniles aged 5 to 10 days, the gonad located on the right side of the dorsal abdominal cavity and the germ cells was significantly larger than the surrounding somatic cells. Ten days after hatching, sexual differences between females and males became obvious, and germline cysts derived from oogonium division were present in the gonad. At 50 days after hatching, ovarian cavities and mature sperm were observed. Moreover, during the study we also observed the coexistence of two sexual characteristics during a male's development. Together, these observations fill current gaps in developmental and genetic biology that will allow a more efficient use of Medaka as an experimental model.