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Characterization, management and protection of biodiversity are the most prominent challenges in conservation biology. Analyses on molecular similarity (Random Amplified Polymorphic DNA-Polymerase Chain Reaction, RAPD-PCR) and morphologic patterns (morphometric and meristic characters) were performed to evaluate the existence of population structuring in the Doce river basin, in a widespread small-sized characin, Astyanax bimaculatus. Three hundred (300) individuals were collected from six locations isolated by waterfalls or dams, in the Doce river basin, Minas Gerais State, Brazil. Genetic differentiation among tributaries was significant ( p<0.00001) and accounted for 21% of total variance ( p<0.00001). Only the Grande waterfall of the Casca river was an effective factor for differentiation of populations ( p<0.00001), and meristic characters were also consistent with molecular data. We concluded that genetic and morphologic variation of this species was not necessarily associated with waterfalls and that molecular and meristic data are effective predictors of population divergence in this basin. The consequences of these findings for the protection and management of aquatic biodiversity were discussed. [ABSTRACT FROM AUTHOR]