Abstract<br> Genome replication is temporally regulated during S phase, with specific genomic regions replicating at defined times in a process that is known as replication timing (RT). Based on 3D cytology in replicating nuclei, we previously proposed a model in which maize euchromatin is subdivided into subcompartments distinguished by chromatin condensation and RT. However, whether this compartmentalization reflects a general nuclear architecture that persists throughout the cell cycle was unclear. To test this model, we conducted two orthogonal assays—Hi-C for genome-wide interaction data and 3D FISH for direct visualization of chromatin organization in maize (Zea mays L.). Hi-C analyses revealed distinct patterns of early-S regions exhibited negative insulation scores with long-range contacts, whereas middle-S regions showed the opposite. Early-S regions showed the strongest correlation with epigenomic signatures of open, transcriptionally active chromatin. 3D oligo FISH painting confirmed that early-S and middle-S replicating regions occupy adjacent but largely non-overlapping nucleoplasmic sub-territories throughout interphase stages, including G1. Together, our findings redefine the maize euchromatin “A” compartment as two spatially distinct subcompartments derived from high-frequency RT transitions between early and middle S along the linear genome. These findings have implications for chromatin-templated processes and underscore the importance of RT as a defining feature of genome organization.