The natterjack toad Bufo calamita is rare in Britain, which is at the northwestern edge of its biogeographical range. We investigated the level of genetic differentiation amongst almost all (34 out of 38) of the surviving British populations of this species, and among six new populations established by translocations during the 1980s. For eight microsatellite loci, allele sizes and frequencies were analysed using samples from each of these populations. The populations clustered into three robustly differentiated groups, each of which corresponded with a geographical region (east/southeast England, Merseyside and Cumbria). The Cumbrian populations showed a further weak geographical substructuring into northern and southern clades. The populations in south Cumbria were genetically more diverse than those in any of the other regions, as judged by the mean numbers of alleles per locus and the mean heterozygosity estimates. The translocated populations clustered close to their founders and, with one exception, did not differ significantly with respect to mean allele numbers, heterozygosity or polymorphism level. However, significant genetic differentiation (as measured by unbiased RST) was found between all but one of the founder-translocation pairs. The implications of this phylogeographic study for the future conservation of B. calamita in Britain are discussed.