Stem cells can divide asymmetrically, generating a daughter stem cell and a cell that is committed for differentiation. Whether mammalian stem cells segregate organelles asymmetrically during cell division remains unclear. Katajisto et al. now show that daughter cells with stem cell properties inherit fewer 'old' mitochondria, indicating that asymmetric partitioning of mitochondria is important for the maintenance of stemness. Mitochondria were labelled (by marking the inner or outer mitochondrial membranes with photoactivatable GFP or SNAP-tag) in cultured human mammary epithelial cells that contain stem-like cells (SLCs). Old mitochondria (aged 10 hours or more) segregated asymmetrically in SLCs, as one daughter cell inherited ∼5.6-fold more old mitochondria than the other. Daughter cells with more old mitochondria differentiated, whereas those with fewer old mitochondria remained SLCs. Asymmetric partitioning required the confinement of old mitochondria to the perinuclear region of the mother cell, and this was dependent on mitochondrial fission.