Populations of the amphibian Bufo calamita were sampled for genetic analysis in eleven areas distributed across its biogeographical range in Europe. Genetic diversity estimates across eight microsatellite loci showed a decline in polymorphism, numbers of alleles and heterozygosity as a function of distance from the presumed ice-age refugium in Iberia. Trials with a selection of tree-building algorithms indicated mat UPGMA of Cavalli-Sforza chord distances (Dc) generated the tree topology most easily reconciled with other biogeographical information. Genetic distance measures were also calibrated against a postglacial event from which the separation of extant populations could be estimated in real time. Dc again outperformed two other measures (Nei's standard distance, Ds, and δμ2) in producing realistic correlations with minimal variance. The genetic analysis was consistent wim die hypodiesis that B. calamita survived in a single refugium (Iberia) during the Pleistocene glaciation and indicated that it spread north and east from there during the last interstadial which commenced about 14 000 years before present (BP). Microsatellites should provide useful tools for biogeographical investigations of other species, especially with respect to patterns of population dispersal.