Three dimensional evaluation of internal structures of small bones with histological methods or conventional X-ray is difficult. Micro-computed tomography (µCT) can provide 3D datasets with high resolution. In this study we applied µCT to analyse tail vertebra of autotomizing lizards. Autotomy is an ability of several reptile families to loose their tail at predicted break points (autotomy plane) to escape from enemies. Until now, these autotomy planes have been described with histological and conventional radiographic methods only. Micro-CT produced high resolution (15 µm voxel size) images clearly showing the autotomy plane in the 3D reconstructions as well as in 2D slices. Micro-CT proved to be of high value for the examination of the 3D position of autotomy planes in tail vertebra of different reptile species. Therefore, µCT will also be useful for the evaluation of 3D bone microarchitecture in other biological research.