Chromosome length is constrained by spindle scaling to ensure faithful mitosis in mammals.

Why eukaryotic genomes are universally divided among multiple chromosomes remains an unresolved question. Although yeast and mouse cells can tolerate chromosomal fusions without impairing viability, we show here that chromosome length in mammalian cells is constrained by a biophysical limit governed by spindle geometry. Using engineered mouse cells carrying fused chromosomes of defined sizes, we identify ~308 Mb as the maximal length tolerated for faithful mitosis. Chromosomes exceeding this threshold disrupt segregation, leading to daughter cell re-coalescence and polyploidization. Aurora B kinase regulates this process by modulating spindle elongation; its inhibition induces mitotic failure even in chromosome configurations within the tolerated threshold of ~308 Mb. These findings explain the structural basis for genome fragmentation in animals and reveal a general mechanism linking chromosome size, spindle dynamics, and genome stability.