This report presents the results of a broad assessment of freshwater diversity in the Douro river basin in Spain and Portugal. The Douro river basin is the largest of the Iberian Peninsula, and an ecological region of great value due to the high diversity of ecosystems and the number of species. A total of 14 sites, either identified as Key Biodiversity Areas (KBAs) or potential KBA sites, were surveyed for fish and macroinvertebrate species. More than 120 species were detected, with more than 10 species listed as threatened with extinction on the IUCN Red List of Threatened Species^TM.