Given the increasing threats to biodiversity and limited resources for conservation, our knowledge about the uncertainty in surrogates for representing comprehensively the spatial conservation priorities for biodiversity, needs to be improved. We present a comprehensive spatial conservation approach for Neotropical biodiversity by including surrogates for three biodiversity attributes: composition (8563 species), structure (663 ecosystems), and function (5382 ecological groups). We evaluated the differences in the representativeness and surrogacy-level of resulting portfolios for each attribute, considering the differences for current established conservation areas (CAs), prioritized areas (PRAs; those selected to complement the CAs), and total areas (TAs: CAs + PRAs). The assessment included the entire Neotropics, and a regionalization approach using the Global 200 Ecoregions by country. Finally, we identified critical areas for conservation based on the coincidence of irreplaceable PRAs among biodiversity attributes. Our results confirm the premise that no single surrogate represents biodiversity comprehensively, providing quantitative evidence to support the importance of using integrative information of surrogates for different levels of biodiversity into identifying priority areas for conservation. The spatial mismatch in the portfolios of areas shows how the use of a single level of biodiversity would lead to the omission of conservation priorities for other levels. We also identified critical areas for conservation where irreplaceable spatial priorities of the different biodiversity attributes matched. These areas coincided with known critical and threatened global biodiversity hotspots, and are mostly located in the Chaco, the Atlantic Forest, the Pantanal, Cerrado, and Caatinga regions, and the moist and dry forests of the northern Andes and Mesoamerica.