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The aim of this study was to analyze the morphological variation of brown trout (Salmo trutta) in the Duero basin, an Atlantic river basin in the Iberian Peninsula, where a spatial segregation of two divergent lineages was previously reported, based on isozyme, microsatellite and mtDNA data. In these studies, two divergent pure regions (Pisuerga and Lower-course) and several hybrid populations between them were identified. Morphological variation was evaluated in 11 populations representative of the genetic differentiation previously observed in the Duero basin, using multivariate analysis on 12 morphometric and 4 meristic traits. A large differentiation between populations was observed (interpopulation component of variance: 41.8%), similar to that previously detected with allozymes and microsatellites. Morphometric differentiation was also reflected by the high classification success of pure and hybrid individuals to their respective populations, using multivariate discriminant functions (94.1% and 79.0%, respectively). All multivariate and clustering analyses performed demonstrated a strong differentiation between the pure regions. The hybrid populations, though showing large differentiation among them, evidenced an intermediate position between the pure samples. Head and body shape traits were the most discriminant among the morphometric characters, while pectoral rays and gillrakers were the most discriminant among the meristic traits. These results confirmed the high divergence of the brown trout from the Duero basin and suggest some traits on which selection could be acting to explain the spatial segregation observed.