Aquatic organisms living in a range of photic environments require specific mechanisms to tune their visual pigments. Maximum absorbance (lambda(max)) of retinal rods in populations of the marine demersal sand goby, (Pomatoschistus minutus; Gobiidae, Teleostei) correlates with the local optic environment. It has been shown that this is not regulated through a physiological response by exchanging the rhodopsin chromophore. To test for evolutionary adaptation, the sequence of the rhodopsin (RH1) gene was analysed in 165 Pomatoschistus minutus individuals from seven populations across its distribution range. Analysis showed a high level of intraspecific polymorphism at the RH1 gene, including nonsynonymous mutations on amino acids, known as spectral tuning sites. Population differentiation at these sites was in agreement with the observed differentiation in lambda(max) values. Analyses of d(N)/d(S) substitution rate ratios and likelihood ratio tests under site-specific models detected a significant signal of positive Darwinian selection on the RH1 gene. A strong discrepancy in differentiation was noticed between RH1 gene variation and the presumably neutral microsatellites and mitochondrial data. Samples did not cluster according to geographical or historical proximity with regards to RH1, but according to the general photic conditions of the habitat environment of the sand goby. This study highlights the usefulness of sensory genes, like rhodopsin, for studying the characteristics of local adaptation in marine nonmodel organisms.