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Conserving freshwater habitats and their biodiversity in the Amazon Basin is a growing challenge in the face of rapid anthropogenic changes. We used the most comprehensive fish‐occurrence database available (2355 valid species; 21,248 sampling points) and 3 ecological criteria (irreplaceability, representativeness, and vulnerability) to identify biodiversity hotspots based on 6 conservation templates (3 proactive, 1 reactive, 1 representative, and 1 balanced) to provide a set of alternative planning solutions for freshwater fish protection in the Amazon Basin. We identified empirically for each template the 17% of sub‐basins that should be conserved and performed a prioritization analysis by identifying current and future (2050) threats (i.e., degree of deforestation and habitat fragmentation by dams). Two of our 3 proactive templates had around 65% of their surface covered by protected areas; high levels of irreplaceability (60% of endemics) and representativeness (71% of the Amazonian fish fauna); and low current and future vulnerability. These 2 templates, then, seemed more robust for conservation prioritization. The future of the selected sub‐basins in these 2 proactive templates is not immediately threatened by human activities, and these sub‐basins host the largest part of Amazonian biodiversity. They could easily be conserved if no additional threats occur between now and 2050. Article Impact Statement: Alternative scenarios of diversity hotspots for freshwater fish can be used to define conservation priorities in the Amazon Basin. [ABSTRACT FROM AUTHOR]