Chromatin located in the cell nucleus is folded into a hierarchy of structures of increasing complexity, from nucleosomes and chromatin fibres to chromatin loops, domains, chromatin compartments and chromosome territories. Its flexible spatial arrangement must ensure that the correct gene expression programs are executed at the right time and in the right cell type and that the chromatin can be rapidly reorganized into highly condensed structures – metaphase chromosomes. The elucidation of these processes is facilitated by chromosome conformation capture (3C) techniques, especially Hi-C, which provides genome-wide information about chromatin contacts in the three-dimensional space. We applied Hi-C to study spatial genome organisation in barley – a model for large-genome cereals. First, we combined Hi-C with flow sorting of metaphase chromosomes and nuclei at G1, S and G2 phase, respectively, and analysed chromatin dynamics during the cell cycle. We observed surprisingly small changes in higher-order chromatin structure between chromosomes and interphase nuclei isolated from root tips but a striking difference between G1 from root-tip and leaf tissue, indicating a different chromatin arrangement in the interphase between rapidly cycling and non-cycling cells. Furthermore, we used the Hi-C data to delineate active and silenced chromatin compartments in the barley genome and provided evidence for their impact on gene expression. Last, we combined barley interactome data with several epigenomic datasets to identify functional chromatin interactions. This allowed us to predict 1754 distal cis-regulatory elements (enhancers or silencers) and their target genes, which will be further validated by reporter assays and bioinformatics approaches. This research was supported by the Czech Science Foundation, award number 21-18794S, and from the project TowArds Next GENeration Crops, reg. no. CZ.02.01.01/00/22_008/0004581 of the ERDF Programme Johannes Amos Comenius.