To investigate the phylogenetic relationships and geographical structure among brown trout S. trutta L. populations from the South Adriatic‐Ionian and Aegean sea basins, mitochondrial DNA nucleotide sequence comparisons were used. A 310‐base‐pair (bp) segment of the control region (D‐loop), and an additional 280‐bp segment of the cytochrome b gene were sequenced from representatives of 13 brown trout populations. Phylogenetic analyses, conducted after combining the data presented with published data from other Eurasian brown trout, revealed four major phylogenetic groups, three of which were found widely distributed within the southern Balkan region. The phylogeographical patterns revealed by mtDNA represent one of the few cases where phylogenetic discontinuity in a gene tree exists without obvious geographical localization within a species' range and has most likely resulted from the differentiation of the major mtDNA clades during Messinian or early Pleistocene times. Finally, the genetic relationships among the populations suggested by mtDNA were generally not in accordance with either allozyme or morphological data.