The biodiversity hotspots are 35 biogeographical regions that have both exceptional endemism and extreme threats to their vegetation integrity, and as such are global conservation priorities. Nonetheless, prior estimates of natural intact vegetation (NIV) in the hotspots are generally imprecise, indirect, coarse, and/or dated. Using moderate- and high-resolution satellite imagery as well as maps of roads, settlements, and fires, we estimate the current extent of NIV for the hotspots. Our analysis indicates that hotspots retain 14.9% of their total area as NIV (~3,546,975 km2). Most hotspots have much less NIV than previously estimated, with half now having 10% NIV by area, a threshold beneath which mean NIV patch area declines precipitously below 1000 ha. Hotspots with the greatest previous NIV estimates suffered the greatest apparent losses. The paucity of NIV is most pronounced in biomes dominated by dry forests, open woodlands, and grasslands, reflecting their historic affinities with agriculture, such that NIV tends to concentrate in select biomes. Low and declining levels of NIV in the hotspots underscore the need for an urgent focus of limited conservation resources on these biologically crucial regions.