Determining how chromosomes are positioned and folded within the nucleus is critical to understanding the role of chromatin topology in gene regulation. Several methods are available for studying chromosome architecture, each with different strengths and limitations. Established imaging approaches and proximity ligation-based chromosome conformation capture (3C) techniques (such as DNA-FISH and Hi-C, respectively) have revealed the existence of chromosome territories, functional nuclear landmarks (such as splicing speckles and the nuclear lamina) and topologically associating domains. Improvements to these methods and the recent development of ligation-free approaches, including GAM, SPRITE and ChIA-Drop, are now helping to uncover new aspects of 3D genome topology that confirm the nucleus to be a complex, highly organized organelle.