The study investigates the three-dimensional organization of human mitotic chromosomes using chromosome conformation capture methods, 5C and Hi-C, across the cell cycle. It reveals two alternative folding states: a compartmentalized, cell-type-specific organization in interphase and a homogeneous, locus-independent state in metaphase. Metaphase chromosomes are found to be organized in a linearly compressed array of consecutive chromatin loops, which is inconsistent with classic hierarchical models. Polymer simulations show that metaphase Hi-C data best fit a linearly-organized structure, suggesting a two-stage process for mitotic chromosome organization: linear compaction by consecutive chromatin loops, followed by axial compression. The study shows that in metaphase, the compartmentalization and topologically associating domains (TADs) characteristic of interphase are lost. Chromosomes in metaphase display a uniform interaction pattern across all cell types, indicating a universal mitotic conformation. The contact probability (P(s)) for mitotic chromosomes decreases slowly from 100 Kb to 10 Mb, followed by a rapid fall-off, suggesting a linear organization of chromatin above 10 Mb. This is consistent with a model of a fractal globule, but with intermediate spatial mixing. Polymer models of mitotic chromosomes were tested, and the best model involved consecutive loops of 80–120 Kb, with linear organization of loci separated by more than 10 Mb. These models showed good agreement with experimental data. The study proposes a two-stage process for mitotic chromosome folding: first, linear compaction of the chromatin fiber into an array of consecutive loops, followed by axial compression. This model explains the observed structure and function of mitotic chromosomes, and suggests that higher-order chromatin structures are re-established in early G1, without carrying epigenetic memory. The findings provide insights into the structural and functional organization of mitotic chromosomes and the mechanisms underlying their condensation.The study investigates the three-dimensional organization of human mitotic chromosomes using chromosome conformation capture methods, 5C and Hi-C, across the cell cycle. It reveals two alternative folding states: a compartmentalized, cell-type-specific organization in interphase and a homogeneous, locus-independent state in metaphase. Metaphase chromosomes are found to be organized in a linearly compressed array of consecutive chromatin loops, which is inconsistent with classic hierarchical models. Polymer simulations show that metaphase Hi-C data best fit a linearly-organized structure, suggesting a two-stage process for mitotic chromosome organization: linear compaction by consecutive chromatin loops, followed by axial compression. The study shows that in metaphase, the compartmentalization and topologically associating domains (TADs) characteristic of interphase are lost. Chromosomes in metaphase display a uniform interaction pattern across all cell types, indicating a universal mitotic conformation. The contact probability (P(s)) for mitotic chromosomes decreases slowly from 100 Kb to 10 Mb, followed by a rapid fall-off, suggesting a linear organization of chromatin above 10 Mb. This is consistent with a model of a fractal globule, but with intermediate spatial mixing. Polymer models of mitotic chromosomes were tested, and the best model involved consecutive loops of 80–120 Kb, with linear organization of loci separated by more than 10 Mb. These models showed good agreement with experimental data. The study proposes a two-stage process for mitotic chromosome folding: first, linear compaction of the chromatin fiber into an array of consecutive loops, followed by axial compression. This model explains the observed structure and function of mitotic chromosomes, and suggests that higher-order chromatin structures are re-established in early G1, without carrying epigenetic memory. The findings provide insights into the structural and functional organization of mitotic chromosomes and the mechanisms underlying their condensation.