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Abstract Global changes, from habitat loss and invasive species to anthropogenic climate change, have initiated the sixth great mass extinction event in Earth's history. As species become threatened and vanish, so too do the broader ecosystems and myriad benefits to human well-being that depend upon biodiversity. Bringing an end to global biodiversity loss requires that limited available resources be guided to those regions that need it most. The biodiversity hotspots do this based on the conservation planning principles of irreplaceability and vulnerability. Here, we review the development of the hotspots over the past two decades and present an analysis of their biodiversity, updated to the current set of 35 regions. We then discuss past and future efforts needed to conserve them, sustaining their fundamental role both as the home of a substantial fraction of global biodiversity and as the ultimate source of many ecosystem services upon which humanity depends.