The relationship between genotype and phenotype is nontrivial due to often complex molecular pathways that make it difficult to unambiguously relate phenotypes to specific genotypes. Photopigments present an opportunity to directly relate the amino acid sequence to the phenotype in the form of the absorbance peak or λmax. We examined the relationship of genotype and phenotype of the invertebrate non-visual opsin retinochrome by conducting reciprocal mutagenesis experiments between the common bay scallop, Argopecten irradians, and the closely related king scallop, Pecten maximus. We identified three amino acid sites of likely functional importance, expressed site-directed mutants in-vitro, and spectrally characterized the resulting phenotype of each mutant photopigment. Our results show that the mutation of amino acids lining the binding pocket of retinochrome can change peak absorbance and may be important for fine spectral tuning; however, residue changes were not always additive in the mutant phenotype. We observed most mutations causing a blue shift in absorbance, with shifts ranging from a 12nm blue shift to 5nm red shift from the wild-type retinochromes. These findings highlight the spectral tuning effect of amino acids away from the Schiff-base linkage site and the counter-ion, and suggest that secondary sites may be responsible for intramolecular epistasis. This work highlights the potential importance of binding pocket shape in the evolution of spectral tuning and builds on our ability to relate genotypic changes to phenotypes in an emerging model for opsin functional analysis.