Freshwater ecosystems are among the most biodiverse yet severely threatened ecosystems in the world. The Mediterranean Basin is a biodiversity hotspot for freshwater biodiversity and lacustrine species inhabiting ancient lakes. Key Biodiversity Areas (KBAs) represent by far the largest network of important sites for biodiversity that have been systematically identified using standardized quantitative criteria, contributing to the global persistence of biodiversity. In this chapter, we compiled a spatial dataset containing the global range maps of 793 freshwater fish mollusks, plants, insects, crustaceans, fungi, and worm species from the IUCN Red List of Threatened Species. Data were collected for the 50 largest natural lakes in the Mediterranean region. We tested for the first time, KBA Criteria to 169 sub-catchments, in order to identify potential KBAs (pKBAs). We determined the threats posed to the species that trigger KBA criteria, alongside the coverage of pKBAs from protected areas. One hundred and forty-four sites (85%) hold 227 species that trigger KBA Criteria (28%) in total. In addition, 78% of the species triggering KBA criteria are found in the Balkans. Only 36% of pKBAs is protected, with the lowest protection found in Turkey and Levante (6%). Our results highlight the importance of undertaking comprehensive KBA assessments for freshwater species and addressing the threats posed to freshwater biodiversity in policies and frameworks, while providing the areas that Mediterranean countries should prioritize when expanding protected area networks to achieve the 30% protected area target by 2030 of the Kunming—Montreal Biodiversity Framework.