Our understanding of the organization of the chromatin fiber within the cell nucleus has made great progress in the last few years. High-resolution techniques based on next-generation sequencing as well as optical imaging that can investigate chromatin conformations down to the single cell level have revealed that chromatin structure is highly heterogeneous at the level of the individual allele. While TAD boundaries and enhancer–promoter pairs emerge as hotspots of 3D proximity, the spatiotemporal dynamics of these different types of chromatin contacts remain largely unexplored. Investigation of chromatin contacts in live single cells is necessary to close this knowledge gap and further enhance the current models of 3D genome organization and enhancer–promoter communication. In this review, we first discuss the potential of single locus labeling to study architectural and enhancer–promoter contacts and provide an overview of the available single locus labeling techniques such as FROS, TALE, CRISPR–dCas9 and ANCHOR, and discuss the latest developments and applications of these systems.