For most species in the Western Palaearctic region, southern Mediterranean peninsulas have been identified as major Quaternary refugia and hotspots of intraspecific diversity, and thus, as areas of particular relevance for the conservation of the evolutionary potential. We analysed the patterns of geographical variation among 26 populations of the Italian stream frog, using both nuclear (allozymes) and mitochondrial (partial cytochrome b sequences) markers. Phylogenetic, phylogeographical and population genetic analyses suggested that the species survived the last glacial-interglacial cycles in two distinct refugia, one restricted to the tip of the Calabrian peninsula, at the extreme south of the species' range, the other spanning from central Calabria to central Apennines and showing evidences for further population subdivision therein. Historical demographic tests suggested a significant population expansion from the latter, which most likely began around the last pleniglacial. This expansion would have led to the rapid colonization of the northern Apennines to the north, and to a secondary contact and population admixture with the population from the southern refugium in southern central Calabria. A comparison of the evolutionary history inferred for the Italian stream frog with the data emerging for other codistributed species suggests: (i) the generality of a multiple-refugia scenario for the Italian peninsula, (ii) the possible occurrence of at least one suture zone in southern Italy, and (iii) that for most species, this Pleistocene refugium is not only a hotspot, but also a melting pot of intraspecific genetic diversity. Finally, the conservation implications of these results are also briefly highlighted.