Smoltification is a metamorphic event in salmon life history, which initiates downstream migration and pre-adapts juvenile salmon for seawater entry. While a number of reports concern thyroid hormones and smoltification, few and inconclusive studies have addressed the potential role of thyrotropin (TSH). TSH is composed of a α-subunit common to gonadotropins, and a β-subunit conferring hormone specificity. We report the presence and functional divergence of duplicated TSH β-subunit paralogs (tshβa and tshβb) in Atlantic salmon. Phylogeny and synteny analyses allowed us to infer that they originated from teleost-specific whole genome duplication. Expression profiles of both paralogs in the pituitary were measured by qPCR throughout smoltification in Atlantic salmon from the endangered Loire-Allier population raised at the Conservatoire National du Saumon Sauvage. This revealed a striking peak of tshβb expression in April, concomitant with downstream migration initiation, while tshβa expression remained relatively constant. In situ hybridization showed two distinct pituitary cell populations, tshβa cells in the anterior adenohypophysis, and tshβb cells near to the pituitary stalk, a location comparable to the pars tuberalis TSH cells involved in seasonal physiology and behaviour in birds and mammals. Functional divergence of tshβ paralogs in Atlantic salmon supports a specific role of tshβb in smoltification.