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The ecological integrity of lotic ecosystems is intimately linked to the quality of their catchments. Environmental protection efforts should therefore be implemented at the relevant catchment-scale in order to support river biodiversity and associated ecosystem services. Therefore, freshwater management can greatly benefit from tools that enable spatially-explicit predictions of water quality across heterogeneous landscapes. To this end, we investigated the relationships between the biological quality of running waters (expressed by the Italian Extended Biotic Index, EBI) and surrounding topographic and land cover matrices in central Italy. To do so, we adopted a spatial approach based on generalized linear models (GLM) that allowed us to identify the catchment features influencing the EBI values, and to produce a map of expected water quality in the region. A gap-analysis was then performed to assess the overlap between the existing regional protected areas and the modelled water quality map. Our findings provide suitable support for the identification of certain land uses in order to preserve riverine biodiversity and hopefully restore lotic ecosystems in Mediterranean catchments.