A biochemical‐genetic analysis was carried out on five samples of three species ofLeuciscus, to verify the genetic divergence between PadanianL. cephalusandL. souffiaand to unravel the taxonomic status of aLeuciscuspopulation preliminarly reported asLeuciscussp., collected from the Adriatic drainage basins and morphologically related toL. lucumonis, a recently described taxon of the Tyrrhenian basins. Both isozymic data on 25 loci and total protein analyses identifiedLeuciscussp. asL. lucumonisand showed the high genetic divergence and polymorphism ofL. souffiawith respect toL. cephalusandL. lucumonis. This study confirms thatL. lucumonisis a valid species that inhabits also Adriatic drainage basins and provides conclusive evidence that it is not originated by hybridization. In addition, genetic data fully support the hypothesis of a sharp divergence ofL. souffiafrom congeneric taxa.