The genetic differentiation and phylogenetic relationships of 13 populations (11 from Greece, one from Albania and one from France) of brown trout (Salmo trutta L.) were investigated at the mtDNA level. RFLP analysis of four segments of mitochondrial DNA (D-loop, cytochrome b and ND-5/6) amplified by PCR was used. Seven of 14 restriction endonucleases were found to detect variability in the ND-5/6 regions, whereas four and two out of 17 and 16 were polymorphic in the D-loop and cytochrome b, respectively. Eleven different haplotypes were observed. The observed interpopulation diversity was very high (mean= 1.65 per cent), whereas the intrapopulation diversity was low in most cases (mean= 0.063 per cent). Five phylogenetic assemblages were identified. The results demonstrate that Greece is one of the regions where brown trout display very high levels of genetic diversity. Most populations were genetically very distinct, possessing private mtDNA genotypes. Therefore, they represent unique gene pools which may warrant individual recognition for conservation and management. The genetic relationships among populations suggested by mtDNA data were not in accordance with allozyme data. This study illustrates the importance of considering the historical biogeography of a species in order to understand its population genetic structure. It also reinforces the view that mtDNA analysis represents a powerful tool to study past and present demographic phenomena.