: One of the important factor that is seems to be involved in sex determination of TSD animal is Sox9. Sox9 has an important role in the molecular sex-determining cascade. When the level of Sox9 protein reach thetreshold, it could drive the bipotential gonad to a testis differentiation . The research is conducted to examine Sox9 expression level in the early stage of gonadal development of Chelonia mydas, a species with TSD. Eggs were incubated in FPT (31±1°C) incubator to produce 100% of female embryo, and the remaining in MPT (26±1°C) produce 100% of male embryo. The examination level expression of mRNA was performed since preTSP till the end of TSP period. Quantification of Sf1 level expression from adrenal-gonad-kidney (AGK) complex of embryonic stage 12, 14, 16 (pre TSP), 17, 19 dan 22 (TSP), incubated in FPT (31±1°C) or MPT (26±1°C) were performed using RT-PCR method. Level expression of housekeeping gene â-actin is used as a control. Amplification product was electrophorized on TAE-Agarose gel and then documenting by using digital camera. The density of each DNA band were examined by using ScionImage Software. The average of 10 scores for each band was convert into a line diagram. RT-PCR result showed that the incubation temperature influenced mRNA expression level of Sox9. Expression level of Sox9 is higher in embryo which were incubated in MPT (26±1°C) than in FPT (31±1°C). Differential expression level of the geneappear from the middle of preTSP till the middle of TSP. The highest level of the difference appear soon before Thermosensitive Period. Differential expression of Sox9 during the stage showed that Sox9 has an important roleduring the early stage of sex determination gonad of Chelonia mydas
