The vertebrate brain shows an extremely conserved layout across taxa. Still, the relative sizes of separate brain regions vary markedly between species. One interesting pattern is that larger brains seem associated with increased relative sizes only of certain brain regions, for instance telencephalon and cerebellum. Till now, the evolutionary association between separate brain regions and overall brain size is based on comparative evidence and remains experimentally untested. Here we test the evolutionary response of brain regions to directional selection on brain size in guppies (Poecilia reticulata) selected for large and small relative brain size. In these animals, artificial selection led to a fast response in relative brain size, while body size remained unchanged. We use micro-CT to investigate how the volumes of 11 main brain regions respond to selection for larger vs. smaller brains. We found no differences in relative brain region volumes between large- and small-brained animals and only minor sex-specific variation. Also, selection did not change allometric scaling between brain and brain region sizes. Our results suggest that brain regions respond similarly to strong directional selection on relative brain size, which indicates that brain anatomy variation in contemporary species most likely stem from direct selection on key regions. This article is protected by copyright. All rights reserved.