Thyroid-stimulating hormone (TSH) is composed of a specific b subunit and an a subunit that is shared with the two pituitary gonadotropins. The three b subunits derive from a common ancestral gene through two genome duplications (1R and 2R) that took place before the radiation of vertebrates. Analysis of genomic data from phylogenetically relevant species allowed us to identify an additional Tshb subunit-related gene that was generated through 2R. This gene, named Tshb2, present in cartilaginous fish, little skate and elephant shark, and in early lobe-finned fish, coelacanth and lungfish, was lost in ray-finned fish and tetrapods. The absence of a second type of TSH receptor (Tshr) gene in these species suggests that both TSHs act through the same receptor. A novel Tshb sister gene, named Tshb3, was generated through the third genomic duplication (3R) that occurred early in the teleost lineage. Tshb3 is present in most teleost groups but was lostin tedraodontiforms. The 3R also generated a second Tshr, named Tshrb. Interestingly, the new Tshrb was translocated from its original chromosomic position after the emergence of eels and was then maintained in its new position. Tshrb was lost in tetraodontiforms and in ostariophysians including zebrafish although the latter species have two TSHs, suggesting that TSHRb may be dispensable. The tissue distribution of duplicated Tshbs and Tshrs was studied in the European eel. The endocrine thyrotropic function in the eel would be essentially mediated by the classical Tshb and Tshra, which are mainly expressed in the pituitary and thyroid, respectively. Tshb3 and Tshrb showed a similar distribution pattern in the brain, pituitary, ovary and adipose tissue, suggesting a possible paracrine/autocrine mode of action in these non-thyroidal tissues. Further studies will be needed to determine the binding specificity of the two receptors and how these two TSH systems are interrelated.