Pinopsin is the opsin most closely related to vertebrate visual pigments on the phylogenetic tree. This opsin has been discovered among many vertebrates, except mammals and teleosts, and was thought to exclusively function in their brain for extraocular photoreception. Here, we show the possibility that pinopsin also contributes to scotopic vision in some vertebrate species. Pinopsin is distributed in the retina of non-teleost fishes and frogs, especially in their rod photoreceptor cells, in addition to their brain. Moreover, the retinal chromophore of pinopsin exhibits a thermal isomerization rate considerably lower than those of cone visual pigments, but comparable to that of rhodopsin. Therefore, pinopsin can function as a rhodopsin-like visual pigment in the retinas of these lower vertebrates. Since pinopsin diversified before the branching of rhodopsin on the phylogenetic tree, two-step adaptation to scotopic vision would have occurred through the independent acquisition of pinopsin and rhodopsin by the vertebrate lineage.