BackgroundThe adaptation of plants to cold stress involves changes in gene expression profiles that are associated with epigenetic regulation. Although the three-dimensional (3D) genome architecture is considered an important epigenetic regulator, the role of 3D genome organization in the cold stress response remains unclear.ResultsIn this study, we developed high-resolution 3D genomic maps using control and cold-treated leaf tissue of the model plantBrachypodium distachyonusing Hi-C to determine how cold stress affects the 3D genome architecture. We generated ~ 1.5 kb resolution chromatin interaction maps and showed that cold stress disrupts different levels of chromosome organization, including A/B compartment transition, a reduction in chromatin compartmentalization and the size of topologically associating domains (TADs), and loss of long-range chromatin loops. Integrating RNA-seq information, we identified cold-response genes and revealed that transcription was largely unaffected by the A/B compartment transition. The cold-response genes were predominantly localized in compartment A. In contrast, transcriptional changes are required for TAD reorganization. We demonstrated that dynamic TAD events were associated with H3K27me3 and H3K27ac state alterations. Moreover, a loss of chromatin looping, rather than a gain of looping, coincides with alterations in gene expression, indicating that chromatin loop disruption may play a more important role than loop formation in the cold-stress response.ConclusionsOur study highlights the multiscale 3D genome reprogramming that occurs during cold stress and expands our knowledge of the mechanisms underlying transcriptional regulation in response to cold stress in plants.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04269-w.