Oxytocin (OT) and vasopressin (VP), and all related hormones, consist of nine amino acids with cysteine residues in positions 1 and 6 that form a six-amino acid cyclic part, and of a C-terminal glycine in α-amidated form. These neuropeptides are classified into oxytocin and vasopressin families based on the amino acid residue at position 8. OT-like and VP-like peptides are present in every vertebrate species. These peptides are a very ancient family of hormones having representatives in diverse species of invertebrates. Invertebrates have either a vasopressin-family peptide or an oxytocin-family peptide, whereas bony fishes, the ancestors of land vertebrates, have both isotocin and vasotocin. Presently, two evolutionary structural lineages have been proposed: an isotocin-mesotocin-OT line, associated with reproductive functions, and a vasotocin-VP line participating in water homeostasis. The ancestral gene encoding the precursor protein has been present in the animal genome for a period exceeding 500 million years of evolution. The exceptionally high stability of this structure of nine-amino acid peptides during the entire process of evolution suggests very powerful selective pressure, possibly by evolution together with respective receptors and specific processing enzymes. A novel gene with a distinct function and expression appeared during evolution through duplication of an ancestral gene. The synteny and order of genes in the neurohypophysial hormone gene locus are conserved in the lamprey, elephant shark, coelacanth, and tetrapods, but disrupted in teleost fishes presumably due to the rearrangements facilitated by a whole-genome duplication event in the teleost fish ancestor.