To better understand the evolution of red-green color vision in vertebrates, we inferred the amino acid sequences of the ancestral pigments of 11 selected visual pigments: the LWS pigments of cave fish (Astyanax fasciatus), frog (Xenopus laevis), chicken (Gallus gallus), chameleon (Anolis carolinensis), goat (Capra hircus), and human (Homo sapiens);and the MWS pigments of cave fish, gecko (Gekko gekko), mouse (Mus musculus), squirrel (Sciurus carolinensis), and human. We constructed these ancestral pigments by introducing the necessary mutations into contemporary pigments and evaluated their absorption spectra using an in vitro assay. The results show that the common ancestor of vertebrates and most other ancestors had LWS pigments. Multiple regression analyses of ancestral and contemporary MWS and LWS pigments show that single mutations S180A, H197Y, Y277F, T285A, A308S, and double mutations S180A/H197Y shift the lambda(max) of the pigments by -7, -28, -8, -15, -27, and 11 nm, respectively. It is most likely that this "five-sites" rule is the molecular basis of spectral tuning in the MWS and LWS pigments during vertebrate evolution.