From mammals to fish, reproduction is driven by luteinizing hormone (LH) and follicle-stimulating hormone (FSH) temporally secreted from the pituitary gland. Teleost fish are an excellent model for addressing the unique regulation and function of each gonadotropin cell since, unlike mammals, they synthesize and secrete LH and FSH from distinct cells. Only very distant vertebrate classes (such as fish and birds) demonstrate the mono-hormonal strategy, suggesting a potential convergent evolution. Cell-specific transcriptome analysis of double-labeled transgenic tilapia expressing GFP and RFP in LH or FSH cells, respectively, yielded genes specifically enriched in each cell type, revealing differences in hormone regulation, receptor expression, cell signaling, and electrical properties. Each cell type expresses a unique GPCR signature that reveals the direct regulation of metabolic and homeostatic hormones. Comparing these novel transcriptomes to that of rat gonadotrophs revealed conserved genes that might specifically contribute to each gonadotropin activity in mammals, suggesting conserved mechanisms controlling the differential regulation of gonadotropins in vertebrates.