Colonization of empty niches can lead to phenotypic diversification and speciation. However, not all lineages have taken advantage of these ecological opportunities. Many lineages fail to diversify even in the presence of ecological niches, indicating that they may lack physiological abilities to exploit them. Identification of such intrinsic constraints is important to understand patterns of evolutionary diversification. Recently, we found that deficiency in docosahexaenoic acid (DHA), an essential omega-3 fatty acid, can constrain freshwater colonization by marine fishes. Three-spined stickleback are primarily marine, but colonized postglacial freshwater habitats and radiated into diverse ecotypes. By contrast, the closely related Japan Sea stickleback have been unable to colonize freshwater habitats. Our genomic analyses revealed gene duplication of fatty acid desaturase 2 (Fads2), a gene encoding a key enzyme in DHA biosynthesis in three-spined sticklebacks. Overexpression of Fads2 increased their ability to synthesize DHA and survive on freshwater-derived diets. Multiple freshwater ray-finned fishes also show a convergent increase in Fads2 copies. We also exhibited that Fads2 copy number variation could underlie adaptive ecological divergence among multiple freshwater environments such as lake and stream habitats, indicating its key role in freshwater colonization and radiation.