To describe comparative population genetic structure of the Chilean silverside Basilichthys microlepidotus and the catfish Trichomycterus areolatus, four rivers and three sites within each river were investigated by the analysis of haplotype polymorphisms of the mitochondrial Control Region. For both species, analyses revealed significant differentiation among rivers and low differences within rivers. However, the species differ in haplotype composition; individuals of B. microlepidotus shared some haplotypes in all four rivers, while individuals of T. areolatus showed a different haplotype composition in most rivers. This difference may be explained by the different ecological features of the species. Assuming that both silversides and catfish were present before the separation of the rivers, B. microlepidotus migrated after river isolation, probably using coastal water, while T. areolatus has probably never migrated between these rivers. The long times that the studied rivers have been separated should be taken into account in future conservation plans for the freshwater fish of Chile.This work was supported by Fondecyt 11060496 to DV. DV thanks also Grant PFB-23 (CONICYT, Chile) and Grant ICM P05-002. CQR thanks Master CONICYT Grant