We inferred the ancestral amino acid (aa) sequences of the four zebrafish RH2 opsins by likelihood-based Bayesian statistics and reconstituted the ancestral opsins by site-directed mutagenesis. The ancestral pigment (A1) to the four zebrafish RH2 pigments and that (A3) to RH2-3 and RH2-4 showed lambda max at 506 nm, while that (A2) to RH2-1 and RH2-2 a lambda max at 474 nm, indicating that a spectral shift had occurred toward the shorter wavelength on the evolutionary lineages A1 to A2 by 32 nm, A2 to RH2-1 by 7 nm, and A3 to RH2-3 by 18 nm. Pigment chimeras and site-directed mutagenesis revealed a large contribution (~15 nm) of glutamic acid to glutamine substitution at residue 122 (E122Q) to the A1 to A2 and A3 to RH2-3 spectral shifts. However, majority of aa replacements is not at close vicinity of retinal and their spectral effects are individually small and not additive. These results indicate that there can be many different ways to spectral differentiations of visual opsins.