Yusuf, M., Kaneyoshi, K., Fukui, K. et al. Use of 3D imaging for providing insights into high-order structure of mitotic chromosomes. Chromosoma 128, 7–13 (2019). https://doi.org/10.1007/s00412-018-0678-5. The high-order structure of metaphase chromosomes remains still under investigation, especially the 30-nm structure that is still controversial. Advanced 3D imaging has provided useful information for our understanding of this detailed structure. It is evident that new technologies together with improved sample preparations and image analyses should be adequately combined. This mini review highlights 3D imaging used for chromosome analysis so far with future imaging directions also highlighted.