The Guadiana River basin's freshwater fish species richness, endemicity and threatened status (92% of native species are threatened) highlight the need for a large-scale study to identify priority areas for their conservation. One of the most common problems in conservation planning is the assessment of a site's relative value for the conservation of regional biodiversity. Here we used a two-tiered approach, which integrates an assessment of biodiversity loss and the evaluation of conservation value through site-specific measures. These measures based on the reference condition approach introduce the ability to make objective comparisons throughout the Guadiana River basin, thus avoiding a priori target areas. We identified a set of biodiversity priority areas of special conservation significance—which contain rare taxa as well as intact fish communities—because of their outstanding contribution to the basin's biodiversity. The inclusion of complete sub-basins in these priority areas might guarantee an optimal solution in terms of spatial aggregation and connectivity. However, the high spatial fragmentation to which the Guadiana River basin is submitted due to river regulation highlights the necessity of a systematic approach to evaluate the capability of the identified priority areas to maintain the Guadiana's freshwater fish biodiversity.