The visual receptor of rods and cones is a covalent complex of the apoprotein, opsin, and the light-sensitive chromophore, 11-cis-retinal. This pigment must fulfill many functions including photoactivation, spectral tuning, signal transmission, inactivation, and chromophore regeneration. Rod and cone photoreceptors employ distinct families of opsins. Although it is well known that these opsin families provide unique ranges in spectral sensitivity, it is unclear whether the families have additional functional differences. In this study, we use evolutionary trace (ET) analysis of 188 vertebrate opsin sequences to identify functionally important sites in each opsin family. We demonstrate the following results. (1) The available vertebrate opsin sequences produce a definitive description of all five vertebrate opsin families. This is the first demonstration of sequence saturation prior to ET analysis, which we term saturated ET (SET). (2) The cone opsin classes have class-specific sites compared to the rod opsin class. These sites reside in the transmembrane region and tune the spectral sensitivity of each opsin class to its characteristic wavelength range. (3) The cytoplasmic loops, primarily responsible for signal transmission and inactivation, are essentially invariant in rod versus cone opsins. This indicates that the electrophysiological differences between rod and cone photoreceptors cannot be ascribed to differences in the protein interaction regions of the opsins. SET shows that chromophore binding and regeneration are the only aspects of opsin structure likely to have functionally significant differences between rods and cones, whereas excitatory and adaptational properties of the opsin families appear to be functionally invariant.