The knowledge of the diversity, richness, and distribution of tropical organisms are poorly understood, and a plethora of new species are still being described even among groups considered well-known. As a result, this inadequate knowledge of the biodiversity has hampered the species’ conservation. Thus, sampling efforts must be urgently optimized to survey important and unique areas and to better allocated the scarce conservation resources, especially in the tropical and developing countries that harbor much of the world biodiversity. We assessed the most relevant regions in terms of environmental dissimilarity for sampling vertebrates (amphibians, birds, and mammals) in the Pernambuco Endemism Center (PEC), located in Atlantic forest and the most threatened region in South America, where only about 1% of remaining forests are protected. We found that 8–41% of the PEC areas showed high sampling relevance for all vertebrate groups, with the non-coastal areas of the PEC presenting the highest sampling relevance in terms of environmental dissimilarity. For all vertebrate groups, the sites with the highest sampling relevance are threatened by fragmentation, and sampling efforts must be allocated to these areas before they are totally converted into human-modified landscapes.