Micro-computed tomography (CT) is a non-invasive technique that is used to obtain three-dimensional (3D) images of tissue structure in small animals. Compared with extensive mammal studies, few 3D imaging studies of fish have been conducted using micro-CT. An optimized method for imaging fish tissue structure is necessary, because they have adapted to diverse environments via functional and structural specialization. Brains of three species with different sizes and habitats were fixed in 4% paraformaldehyde and immersed in non-ionic iodinated contrast agent (Iopamiron). We examined the relationship between Iopamiron concentration and immersion time to determine universally optimal conditions for use in fish. We reconstructed 3D images of whole fish brains from cross-sections of brains from the Malabar grouper (Epinephelus malabaricus), bastard halibut (Paralichthys olivaceus), and threespot wrasse (Halichoeres trimaculatus). Developmental changes in brain structure were observed in the bastard halibut. Most brain regions of the threespot wrasse were distinguishable, although inner regions of the brain were less visible. Histological techniques are typically used to observe fish brain structure, despite its drawbacks in terms of tissue sample preparation (shrinkage and distortion) and image capture (3D image constriction). The technique examined in the present study solves these problems and allows for the simultaneous handling of multiple specimens. Micro-CT imaging is suitable for observing the surfaces and inner structures of fish of various species. Copyright © 2019 Elsevier B.V. All rights reserved.