Mitochondrial DNA analyses using four restriction enzymes (AvaII, EcoRI, BamHI and XbaI) have been carried out to evaluate stocking success and to assess the amount and distribution of genetic variation among different populations of Spanish brown trout collected in rivers flowing to the Mediterranean and Cantabrian seas, and to the Atlantic Ocean. The hatchery stock was conspicuously different from the wild populations. mtDNA variation provided useful genetic markers to evaluate the success of stocking procedures in Spanish rivers. A relationship was found between population size and mtDNA variation. Between-population haplotype differences found in this work support the hypothesis of a recent divergence of all the Spanish brown trout from a common ancestral genotype.