© Jacinto Román et al.The Doñana Biosphere Reserve is one of the most recognized and studied natural areas in the world, hence the importance of having an openly accessible species checklist. Previous lists are outdated, often have limited taxonomic scope, lack clearly defined inclusion and exclusion criteria, or are practically inaccessible. Here, an updated checklist of vertebrate species for the Doñana Biosphere Reserve based on explicit spatial, temporal, and biological criteria is presented, reviewed and complemented by expert zoologists. The resulting inventory includes 700 vertebrate species, with birds (60%) and fishes (26%) being the most diverse groups. Seven species are considered extinct in the region, two are possibly extinct (i.e., have not been recorded in the past decade), and 32 are classified as exotic. This updated and curated checklist fills a critical gap in regional biodiversity knowledge and establishes a robust foundation for future monitoring, research, and conservation efforts in this ecologically unique and increasingly threatened area of southwestern Europe.