Haematopoietic stem cells (HSCs) are arguably the most extensively characterized tissue stem cells. Since the identification of HSCs by prospective isolation1, complex multi-parameter flow cytometric isolation of phenotypic subsets has facilitated studies on many aspects of HSC biology, including self-renewal2-4, differentiation, ageing, niche5, and diversity6-8. Here we demonstrate by unbiased multi-step screening, identification of a single gene, homeobox B5 (Hoxb5, also known asHox-2.1), with expression in the bone marrow that is limited to long-term (LT)-HSCs in mice. Using a mouse single-colour tri-mCherry reporter driven by endogenousHoxb5regulation, we show that only the Hoxb5+ HSCs exhibit long-term reconstitution capacity after transplantation in primary transplant recipients and, notably, in secondary recipients. Only 7-35% of various previously defined immunophenotypic HSCs are LT-HSCs. Finally, byin situimaging of mouse bone marrow, we show that >94% of LT-HSCs (Hoxb5+) are directly attached to VE-cadherin+ cells, implicating the perivascular space as a near-homogenous location of LT-HSCs.