Background <p>Genome spatial organization plays a fundamental role in biological function across all domains of life. While the principles of nuclear architecture have been well-characterized in animals and plants, their functional relevance in filamentous fungi remains largely uncharacterized. The wheat pathogen <i>Zymoseptoria tritici</i> presents a unique model for genome evolution, with a compartmentalized genome comprising conserved core and highly variable accessory chromosomes linked to genome plasticity. Here, we present the first 3D genome analysis of a eukaryotic organism with an extensive set of accessory chromosomes, revealing a hierarchical genome architecture integrating core and accessory regions.</p> Results <p>At the nuclear level, centromere clustering defines the global genome conformation. Accessory chromosomes are spatially segregated from core arms but maintain focal contacts with pericentromeric regions of core chromosomes, contributing to mitotic stability. At finer resolution, we identify homotypic interactions among heterochromatin-rich compartments and self-interacting domains demarcated by specific histone marks, gene expression profiles, and insulator-like sequence motifs. Notably, a subset of highly insulated, transposon-rich heterochromatic domains forms strong inter-domain interactions. Additionally, domains defined under axenic conditions with coordinated transcriptional activation during wheat infection suggest a link between 3D architecture and dynamic gene regulation.</p> Conclusions <p>Our study uncovers the multi-scale principles of nuclear organization in a major fungal plant pathogen and reveals how hierarchical nuclear architecture contributes to gene expression coordination and genome stability. These findings establish a conceptual framework for investigating 3D genome function and chromatin-mediated regulation in filamentous fungi and other eukaryotic microbes.</p>