We explore the historical demography of the Adriatic lineage of brown trout and more explicitly the colonization and phylogenetic placement of Ohrid trout, based on variation at 12 microsatellite loci and the mtDNA control region. All Adriatic basin haplotypes reside in derived positions in a network that represents the entire lineage. The central presumably most ancestral haplotype in this network is restricted to the Iberian Peninsula, where it is very common, supporting a Western Mediterranean origin for the lineage. The expansion statistic R2, Bayesian based estimates of demographic parameters, and star-like genealogies support expansions on several geographic scales, whereas application of pairwise mismatch analysis was somewhat ambiguous. The estimated time since expansion (155,000 years ago) for the Adriatic lineage was supported by a narrow confidence interval compared to previous studies. Based on microsatellite and mtDNA sequence variation, the endemic Ohrid trout represents a monophyletic lineage isolated from other Adriatic basin populations, but nonetheless most likely evolving from within the Adriatic lineage of brown trout. Our results do not support the existence of population structuring within Lake Ohrid, even though samples included two putative intra-lacustrine forms. In the interests of protecting the unique biodiversity of this ancient ecosystem, we recommend retaining the taxonomic epithet Salmo letnica for the endemic Ohrid trout.