Most studies seeking to provide evolutionary explanations for brain size variability have relied on interspecific comparisons, while intraspecific studies utilizing ecologically divergent populations to this effect are rare. We investigated the brain size and structure of first-generation laboratory-bred nine-spined sticklebacks (Pungitius pungitius) from four geographically and genetically isolated populations originating from markedly different habitats. We found that the relative size of bulbus olfactorius and telencephalon was significantly larger in marine than in pond populations. Significant, but habitat-independent population differences were also found in relative brain and cerebellum sizes. The consistent, habitat-specific differences in the relative size of bulbus olfactorius and telencephalon suggest their adaptive reduction in response to reduced (biotic and abiotic) habitat complexity in pond environments. In general, the results suggest that genetically based brain size and structure differences can evolve relatively rapidly and in repeatable fashion with respect to habitat structure.