The receptors for thyrotropin (TSHR), lutropin/chorionic gonadotropin (LH/CGR) and follitropin (FSHR) belong to the same subfamily of G protein-coupled receptors. The specificity of recognition of their cognate hormone involves a limited number of residues in the leucine-rich repeats present in the N-terminal ectodomain of the receptor. It is admitted that receptors of this subfamily co-evoluted with their respective ligands. The secretion of chorionic gonadotropin is restricted to gestation of primates and equidae. We hypothesized that, facing the challenge of a new hormone, the glycoprotein hormone receptors would have evolved differently in equidae and human so that distinct residues are involved in hormone specificity. In particular, it is known that equine chorionic gonadotropin has a dual (FSH and LH) activity when administered to other species. In the present work, we cloned and characterized functionally the equine TSHR (eTSHR) which shares 89% homology with the human TSHR (hTSHR). The eTSHR is not responsive to equine CG but is more sensitive to human CG (hCG) than the hTSHR. Three residues, at position 60, 229 and 235 of the ectodomain, are responsible for this difference in sensitivity as shown by modelization and targeted mutagenesis followed by in vitro functional characterization. The phylogenetic approach is a suitable approach to identify determinants of specificity of receptors.