The vertebrate retina is a part of the central nervous system (CNS) that can be visualized in a non-invasive manner. The retina is a thin multi-layered sensory tissue of neural cells that contains millions of photoreceptors. There are two physiologically distinct types of photoreceptors across the retina, the cones and the rods. The light sensitivity of the photoreceptors results from the presence of visual pigment molecules in the outer segment of the cone and rod cells. All visual pigments are composed of two components: retinal (vitamin A aldehyde), termed a chromophore, bound to a protein called opsin. The spectral absorption properties of a visual pigment are mainly determined by the amino acid sequence of the opsin protein and the type of chromophore (A₁ or A₂ or mixed) bound to the opsin. There is a shifting from A₁- to A₂-based chromophores in the same opsin due to different environmental factors, e.g. aquatic environments. A₁/A₂ visual pigment ratios play a dominant role in determining rod λ_max in rainbow trout (Oncorhynchus mykiss), shifting the λ_max of the visual pigment and spectral bandwidth of absorbance (HBW, half-band width). The spectral absorption characteristics of the retinal rod photoreceptors in the Rainbow Trout (Oncorhynchus mykiss) were measured using the charge-coupled device (CCD)- based microspectrophotometer (MSP) system. We investigated retina preparations of juvenile rainbow trout (Oncorhynchus mykiss) using 54 rod photoreceptors. The spectral sensitivity of each rod photoreceptor was calculated using λ_max (wavelength of maximum absorbance) and HBW. The mean wavelength of maximum absorbance values for the alpha-bands were 503.5±2.5nm for rhodopsin- dominated rods, 523.1±7.9nm for porphyropsin-dominated rods, and 509.6±1.9nm for rhodopsin porphyropsin mixed rods. The half-band width (HBW) of the main absorption bands was 4567±771 cm-1 for vitamin A₁-based visual pigments and 4697±487 cm-1 for vitamin A₂-based visual pigments. Our results provide the evidence of shifting the λ_max of the visual pigment to a longer wavelength, broadening spectral bandwidth of absorbance, and presence three fundamentally different classes of rod photoreceptors in the retina of the rainbow trout depending on A₁/A₂ visual pigment ratios due to different environmental variables, e.g. aquatic environments.