The support of pluripotent cells over time is an essential feature of develop-ment. In eutherian embryos, pluripotency is maintained from naïve states inperi-implantation to primed pluripotency at gastrulation. To understand howthese states emerged, we reconstructthe evolutionary trajectory of thePou5gene family, which contains the centralpluripotency factor OCT4. By couplingevolutionary sequence analysis with functional studies in mouse embryonicstem cells, wefind that the ability of POU5 proteins to support pluripotencyoriginated in the gnathostome lineage, prior to the generation of two para-logues,Pou5f1andPou5f3via gene duplication. In osteichthyans, retainingboth genes, the paralogues differ in their support of naïve and primed plur-ipotency. The specialization of these duplicates enables the diversification offunction in self-renewal and differentiation. By integrating sequence evolution,cell phenotypes, developmental contexts and structural modelling, we pin-point OCT4 regions sufficient for naïve pluripotency and describe theiradaptation over evolutionary time.