Site conservation is among the most effective means to reduce global biodiversity loss. Therefore, it is critical to identify those sites where unique biodiversity must be conserved immediately. To this end, the concept of key biodiversity areas (KBAs) has been developed, seeking to identify and, ultimately, ensure that networks of globally important sites are safeguarded. This methodology builds up from the identification of species conservation targets (through the IUCN Red List) and nests within larger-scale conservation approaches. Sites are selected using standardized, globally applicable, threshold-based criteria, driven by the distribution and population of species that require site-level conservation. The criteria address the two key issues for setting site conservation priorities: vulnerability and irreplaceability. We also propose quantitative thresholds for the identification of KBAs meeting each criterion, based on a review of existing approaches and ecological theory to date. However, these thresholds require extensive testing, especially in aquatic systems.