Thyroid-stimulating hormone (TSH) regulates the growth and function of thyroid for formation of thyroid hormones, which are involved in regulation of growth, development, metabolism and reproduction. TSHβ and their thyroidal receptors have been investigated extensively in mammals, but comparative studies in lower vertebrates have been considerably less. Our lab had first reported thyroid stimulating hormone b-subunit (TSHb) expressed in the gonads and pituitary of the orange-spotted grouper (Epinephelus coioides), Further analysis found TSHb expressed in the gonads of the red-spotted grouper (Epinephelus akaara).To elucidate the mechanisms regulating TSHb expression, we isolated a 5.1-kb fragment of the red-spotted grouper 5’-flanking region by Genome walking .1.8kb of this upstream region was used to produce GFP reporter construct (pBK-TOL2) for analysis of tissue-specific expression in zebrafish embryos. During 12hpf-30hpf stage, GFP expressed in the PGC; 36hpf-7dpf stage, GFP expressed not only in the PGC but also in pituitary. Western blot analysis showed that GFP protein was detected early in the 10hpf stage embryos. Five sequentially deleted fragments of this upstream region were used to produce GFP reporter constructs (pGL-3BGFP) for analysis of tissue-specific expression in zebrafish embryos. The results also show that a 613-bp upstream fragment is sufficient to direct strong expression of the GFP in gonads but also in pituitary. The Co-localization of TSHb promoter with that of VASA, shown TSHb promoter directed GFP express close to PGC.