In the present study, mitochondrial DNA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was used to assess the phylogenetic and phylogeographic relationships among 27 brown trout Salmo trutta populations from Turkey. The complete NADH 5/6 region and a second segment comprising the cytochrome b gene and D-loop of mtDNA amplified by PCR were digested with six and five restriction enzymes, respectively. A total of 27 haplotypes were observed and divided into three major phylogenetic assemblages, namely Danubian (DA), Adriatic (AD) and a newly proposed Tigris (TI) lineage. The timing of the net nucleotide divergence between the major lineages along with the geological history of Turkey suggested pre-Pleistocene isolation of the Turkish brown trout and provided evidence that Turkey could be considered as a centre of diversification for these lineages. The average haplotype diversity (0(.)1397) and the nucleotide diversity (0(.)000416) within populations were low in comparison to the observed interpopulation nucleotide diversity (0(.)021266). PCR-RFLP analysis showed that most of the mtDNA sequence variation found in the Turkish brown trout populations was imputable to differences among lineages. On the other hand, there was also an obvious relationship between geographical distribution of the populations and their clustering. The present study showed that brown trout populations from Turkey are highly divergent and mainly have a unique genetic profile that could be used for conservation and management purposes. (c) 2006 The Fisheries Society of the British Isles.