Functional traits of river fish assemblages have rarely been studied in the Eastern Mediterranean region. A dataset of 254 Greek electrofished near-natural sites (427 samples) was analyzed to explore native fish functional structure across three spatial scales: (a) among six ecoregions, (b) within a single ecoregion, and (c) in a river basin. Overall, 76 native fish species were assigned eight functional categories, involving 24 different traits. Bipartite networks were used to interpret spatial patterns of species trait data. Two major trait assemblage types were detected in all three spatial scales: the upland type, dominated by insectivorous, benthic, rheophilic, intolerant species, and the lowland type, incorporating omnivorous, limnophilic, phytophilic, and tolerant species. In order to provide further insights between traits and key environmental variables, redundancy analysis (RDA) was employed. The RDA depicted a strong upstream–downstream environmental gradient. Upland and lowland assemblage types form two distinct functional zones along the upstream and downstream river stretches, respectively. Yet, no consistent boundary criteria seem to exist between them. Notably, within a region of high fish assemblage heterogeneity such as the Eastern Mediterranean, functional patterns follow well-established phenomena along the upstream–downstream longitudinal profile of temperate lotic waters.