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Given the multiple stressors affecting freshwater ecosystems and the limited resources devoted to their management, effective conservation of freshwater biodiversity requires regional prioritization. Patagonian wetlands are essential for regional biodiversity and the economy, but they are still far from reaching global conservation targets and many of them could disappear due to climate change. Our study aimed at prioritizing wetlands based on aquatic and terrestrial biodiversity, their conservation status and vulnerability to climate change. First, we identified 43 priority wetlands containing all aquatic biodiversity collected in 82 Patagonian wetlands located over a 1500 km north–south gradient, by using the software Marxan. Then, we ranked within priority wetlands according to their conservation status (low priority if they were already protected; medium priority if not), importance for terrestrial biodiversity conservation (high priority) and vulnerability to climate change. Highly ranked priority wetlands in National Parks (low priority), contained diverse wetlands (57% aquatic taxon richness), including a large proportion of rare species (33%). High priority wetlands are oases of water in an arid and semiarid steppe, containing not only a large proportion of the aquatic biodiversity, but also acting as a refuge for terrestrial flora and fauna. Different management actions are proposed according to wetland priority level (e.g. fencing, creation of artificial ponds), and since 20% of medium priority and 36% of high priority wetlands are expected to disappear by 2050, their inclusion in conservation or restoration plans needs to be carefully evaluated.