The melatonin receptor family is composed of three members, MT(1) and MT(2) receptors that bind melatonin with high affinity and the orphan GPR50 that does not bind melatonin but shares significant sequence homology with the two other subtypes. In the absence of any known ligand for this orphan receptor, little is still known about its function. We recently reported the development of the first anti-GPR50 antibodies that reliably recognized the recombinant human GPR50. We here used these antibodies to study the expression of GPR50 in mouse, rat and human hypothalamus, a region reported to express GPR50 mRNA. GPR50 immunoreactivity (ir) was observed in dorsomedial hypothalamic (DMH) cells co-stained with the neuronal marker HuC/D. GPR50-ir was also observed in cells of the ependymal layer of the third ventricle that co-stained with vimentin. More specifically, its localization in the lower region of the third ventricle and along the long basal processes contacting portal blood vessels in the median eminence (ME) suggested expression of GPR50 in tanycytes. Consistent staining patterns were observed in all three species with two different antibodies. Taken together, our study validates two GPR50-specific antibodies for the use in rodent and human tissue. Evolutionary conserved expression of GPR50 in DMH neurons and tanycytes, together with previously reported expression of the receptor in the pituitary, support the potentially important role of GPR50 in key hypothalamic functions, including regulation of the hypothalamo-pituitary axes.