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Tentatively dated, the Plio-/ Pleistocene origin of the ancient Lake Ohrid on the Balkan Peninsula makes it the oldest ancient lake in Europe. Given the surface area of the lake and the adjusted endemicity rate, it may be also defined as the most diverse of all the ancient lakes in the world. From all the animal groups endemic to this lake, gammarids are amongst the most scarcely known in terms of their diversity and phylogenetic relationships. Partial DNA sequences of two mitochondrial genes, cytochrome oxidase subunit I ( cox1) and 16S ribosomal RNA ( 16 S r RNA) of eight known endemic Gammarus species from the Lake Ohrid valley were analysed. Phylogenetic analyses showed that endemic Gammarus species comprise an ancient species flock, with Gammarus sketi from the feeder springs being their sister taxon outside the lake. Amongst the species inhabiting the lake, Gammarus solidus and Gammarus salemaai are morphologically and molecularly well defined. By contrast, Gammarus ochridensis, Gammarus parechiniformis, Gammarus lychnidensis, and Gammarus stankokaramani revealed high discrepancy between morphological and genetic data. None of these morphospecies form a monophyletic clade and a significant degree of apparent gene flow occurs between them. This could be caused by incomplete lineage sorting and/or hybridization events. Two novel mtDNA lineages were found within the lake, possibly constituting two new species ( Gammarus sp. 1 and Gammarus sp. 2). Molecular clock analysis showed that the split between G. sketi and the Gammarus species flock from the lake occurred approximately 5-7 Mya, whereas within the flock there were at least two intralacustrine radiations: one estimated at 2-3 Mya and the second at less than 1 Mya. The first one could be associated with the origin of the lake and the second with the lake water-level fluctuations during Pleistocene. © 2013 The Linnean Society of London [ABSTRACT FROM AUTHOR]