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Rana temporaria is one of the most widespread Palearctic brown frogs. We aimed to clarify distribution pattern of two main genetic clades in the understudied Balkan peninsula by using 16SrRNA and MT-CYTB sequences, already widely applied in analyses of populations from other parts of Europe, while focusing on the broad area along the Morava river (central Balkans) as a known gap in the species distribution. Additionally, we were interested in revealing the extent of haplotype diversity within the main genetic clades in the Balkans, particularly around the supposed suture zone. The results revealed a suture zone between the Western and Eastern Clades in the central part of the Balkan Peninsula. This indicated the existence of a historical barrier between the Balkan Mountain Belt and geographically close mountains surrounding the Vlasina Plateau (Rhodope/Serbian–Macedonian Massif). The overall observed haplotype diversity in populations of R. temporaria from the Balkan Peninsula seems high. Harboring both main genetic clades of R. temporaria qualifies the Balkan Peninsula as another important center of species’ genetic diversity, as well as rich in unique haplotypes. This points out the necessity of applying conservation measures focused on the common European frog populations and habitats in this part of the species’ distribution area.