Hotspots of genetic diversity are regions of utmost importance for species survival and conservation, and their intimate link with the geographic location of glacial refugia has been well established. Nonetheless, the microevolutionary processes underlying the generation of hotspots in such regions have only recently become a fervent field of research. We investigated the phylogeographic and population genetic structure of the agile frog, Rana dalmatina, within its putative refugium in peninsular Italy. We found this region to harbour far more diversity, phylogeographic structure, and lineages of ancient origin than that by the rest of the species' range in Europe. This pattern appeared to be well explained by climate-driven microevolutionary processes that occurred during both glacial and interglacial epochs. Therefore, the inferred evolutionary history of R. dalmatina in Italy supports a view of glacial refugia as ‘factories' rather than as repositories of genetic diversity, with significant implications for conservation strategies for hotspots.