Tilapia feature asynchronous oocyte development and batch spawning. We conducted this study to furtherunderstand ovarian maturation and the physiological mechanisms involved in non-synchronous spawners, such as tilapia.Changes in sex steroid hormones (estradiol-17β[E2] and progesterone [P]), vitellogenin (Vtg) levels, and VtgmRNA expression were studied during ovarian development in Oreochromis niloticus using histological, enzyme-linked immunoassay, and quantitative reverse transcription-polymerase chain reaction methods, respectively. Theresults showed that the gonadosomatic index (GSI) of O. niloticus paralleled ovarian development and peaked at stage V.Serum E2 levels increased significantly beginning at stage II, peaked at stage IV, then decreased significantly at stage V,whereas P levels increased gradually beginning at stage II, peaked at stage V, and decreased significantly at stage VI.Hepatic Vtg content increased initially, decreased, and then peaked at stage IV. Serum and ovarian Vtg contents increasedgradually beginning at stage II, peaked at stage V, and decreased significantly at stage VI. Hepatic Vtg mRNAexpression peaked at stage III and decreased gradually atstage IV. Ovarian Vtg mRNA expression was relatively lowerand peaked at stage V. These results indicate that E2 and P play different roles during the early and late ovarian developmentalstages, respectively. A close correlation was detected between changes in Vtg content in different tissues andovarian maturation.Vtg mRNA from the liver and ovary were involved in Vtg synthesis; the liver is the major syntheticorgan active during the yolk accumulation phase, but ovarian Vtg synthesis was relatively low.We provide some basicdata to further explore the endocrine regulatory mechanisms in non-synchronous spawning fish.