Aim Freshwater subterranean amphipods with low dispersal abilities are known from both sides of the impermeable barrier, the Adriatic Sea. We tested the hypothesis that historical marine regression–transgression cycles shaped the distribution patterns of subterranean amphipods through repeated cycles of dispersal and vicariance against the hypothesis that subterranean amphipods colonized both sides of the Adriatic Sea independently. Location Western Balkan Peninsula, Adriatic Sea Islands and Apennine Peninsula, Europe. Taxon Genus Niphargus, a clade of freshwater subterranean amphipods (Crustacea: Amphipoda). Methods The taxonomic structure of the studied clade was revised using unilocus species delimitation methods. The timeframe of cladogenetic events was inferred using a multi‐locus time‐calibrated phylogeny and compared to the main regression–transgression events in the Miocene and Pleistocene. The geographical origin of the studied clade, species range expansions and contractions, as well as vicariance events were assessed through modelling of historical biogeography. Results Subterranean amphipods of the genus Niphargus, found on both sides of the Adriatic Sea, form a monophylum. The reconstructions of ancestral ranges suggest that the clade emerged in the Balkan Peninsula, dispersed three times independently to the Apennine Peninsula and once back to the Balkans. Adriatic Islands were colonized multiple times, predominantly from the Balkan Peninsula. The dispersal–vicariance events correspond to historical regression–transgression cycles in Miocene and Pleistocene. Main conclusions Marine regression–transgression cycles apparently shaped the distribution patterns of subterranean amphipods while the alternative hypothesis received no support. The actual distribution of subterranean faunas apparently reflects old biogeographical events.