The visual pigments of rods and cones were studied in eight Fennoscandian populations of nine-spined stickleback (Pungitius pungitius). The wavelength of maximum absorbance (lmax) of the rod pigment varied between populations from 504 to 530 nm due to varying proportions of chromophores A1 and A2 in the same opsin. Four spectral classes of cones were found (two S-cones, M-cones, and L-cones), correlating with the four classes of vertebrate cone pigments. M-cone chromophore proportions were carefully estimated from absorptance spectra and compared with those of rods in the same population. In four populations, spectra of both photoreceptor types indicated A2 dominance (population-mean lmax 525–530 nm for rods and 535–544 nm for M-cones). In the four remaining populations, however, rod spectra (mean lmax 504–511 nm) indicated strong A1 dominance, whereas M-cone spectra (mean lmax 519–534 nm) suggested substantial fractions of A2. Quantitative analysis by three methods confirmed that rods and cones in these populations use significantly different chromophore proportions (P < 0.05). The outcome is a shift of M-cone spectra towards longer wavelengths and a better match to the photic environment (light spectra peaking > 560 nm in all the habitats) than would result from the chromophore proportions of the rods. Chromophore content was also observed to vary partly independently in M- and L-cones with potential consequences for M-L wavelength discrimination. This is the first demonstration that selective processing of chromophore in rods and cones, and in different cone types, may be ecologically relevant.