The guppy shows variation in color vision. Studying the evolutionary forces acting on color vision polymorphism in the guppy is important not only for the understanding the mechanisms explaining the maintenance of this variation, but also for understanding sexual selection and the evolution of highly variable male color patterns and female preference. We determined the sequences of six-opsin genes by analyzing ten populations in Trinidad and Tobago. We examined whether the sequence variation in the six-opsin genes was maintained by natural selection. The results showed that all the examined opsin genes had variation at nonsynonymous sites. The coalescent simulations did not detect significant outliers in the observed Tajima’s D values. Meanwhile FST values of opsin genes were significantly larger than those of the non-opsin reference genes, suggesting that natural selection favors different alleles at the opsin genes in different populations. We suggest that the diversity in opsin genes is maintained by divergent selection among populations. In combination with the considerable migration potential and gene flow of these fish, this may explain the maintenance of the polymorphism within populations.