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Aim: A detailed, comparative DNA‐barcoding and morphospecies‐based overview of the vertical and horizontal distribution of Lake Ohrid's endemic Gammarus species flock is provided. Re‐evaluation of the time frame for the onset of the species flock and identification of events that putatively influenced diversification processes. Location: Lake Ohrid, Balkan Peninsula, Europe. Taxon: Gammarus species flock (Amphipoda, Crustacea). Methods: Extensive sampling and DNA barcoding of 600 individuals was carried out. DNA sequences were analysed using species delimitation methods, haplotype network reconstructions, Bayesian molecular dating and demographic analyses. The COI‐based species delimitation results were validated with nuclear 28S RNA data. Results: The species flock distribution has a weak horizontal but clear vertical structure. The diversity along bathymetric gradients correlates with temperature and salinity; and the highest diversity is observed in sublittoral and springs on the lake's shore. Two new molecular units representing putatively new species are revealed and supported also by the nuclear marker. The onset of flock radiation overlaps with the time of lake formation. The COI gene shows signs of positive selection and an acceleration in substitution rate. The demographic changes within the flock happened during the last thousands years. Main Conclusions: Distribution of the Gammarus species flock is vertically structured, reflecting habitat zonation. Parapatric speciation is one of the possible mechanisms behind the flock's diversification. Detection of new MOTUs suggests that the flock's diversity is still not fully revealed. Simultaneously, failure to collect three previously known MOTUs suggests a loss of gammarid diversity in the lake. This represents, together with the current threats to the lake ecosystem (i.e. climate changes, development of tourism), a clear call for conservation efforts. The speciation events and demographic changes within the flock relate presumably to glacial and postglacial water level changes and to the colonisation of new depth ranges and the associated springs. [ABSTRACT FROM AUTHOR]