In ecosystems with high fish species endemicity, such as Mediterranean-type rivers, biogeographical differences among ecoregions present serious obstacles to developing broadly-applicable river bioassessment indices. This impediment has contributed to a serious time-lag in developing EU policy-relevant fish-based indices in the Eastern Mediterranean countries. Here we present the first model-based fish index for the Eastern Mediterranean (the Hellenic Fish Index, HeFI) in an effort to overcome biogeographic differences among the area's biotically heterogeneous rivers. The index is based on modelled reference conditions and employs site-specific electrofished fish samples from an extensive dataset from Greece that covers six freshwater ecoregions, including five transboundary river basins flowing through six countries. Environmental and anthropogenic pressure data were procured from 403 sampled river sites and ecologically-relevant traits were defined for 103 collected fish species. For the development of the index, we first diagnosed least degraded sites forming a calibrated reference site dataset and secondly quantified differences of fish metrics between the reference and impaired sites. Four trait-based fish metrics showed the best ability to discriminate between impaired and reference sites. The index performed well in discriminating anthropogenic pressure classes, giving a significant negative linear response to a gradient of anthropogenic degradation. HeFI successfully assessed both small and large rivers in different freshwater ecoregions. This geographically broad-scale index development shows that key trait-based reference conditions can be produced by a predictive model in remarkably heterogeneous rivers where range-restricted fishes dominate. This index promotes a screening-level bioassessment application that may be further developed and refined with relevant monitoring.