SummaryMany biomes still lack an overall view of their macro-functional structure (i.e., natural biogeographical regions and zones), including the Atlantic Forest biodiversity hotspot. The effective design of protected areas depends on the spatial identification of units with ecologically distinct content, whether it be floristic, phylogenetic or functional. This study used a regionalization approach to identify the potential functional regions of the Atlantic Forest by interpolating functional data from forest remnants into the entire original occurrence area of the biome, including deforested lands. Conservation status was then estimated. Analysis of seven traits of leaf, wood, seed and plant size revealed that the biome is structured over 14 functional regions and three zones (clusters of regions). Functional regions represented specific combinations of traits rather than being characterized by extremely high or low values of a single trait. They retained an average of 29.5% of forest remnants (range: 7.63–54.66%) and 10.82% of protected areas (range: 0.35–35.78%). By analysing the functional space occupied by all regions, captured by two principal component analysis axes using the pixel-level information contained in interpolated trait maps, we showed that large parts of this space were not covered by forest remnants or protected areas and that the most represented regions had serious deficits in protected areas. Although the Serra do Mar mountain range in the south and south-east Atlantic Forest is relevant as a centre of species endemism and richness and has received considerable attention for carrying out ecological studies and creating protection areas, this range does not fully encompass the functional biodiversity of such a rich biome. Our results demonstrate the potential for combining regionalization and conservation approaches to unravel the macro-structures of biomes.