The European natterjack toad (Bufo calamita) has declined rapidly in recent years, primarily due to loss of habitat, and in Denmark it is estimated that 50% of the isolated populations are lost each decade. To efficiently manage and conserve this species and its genetic diversity, knowledge of the genetic structure is crucial. Based on nine polymorphic microsatellite loci, the genetic diversity, genetic structure and gene flow were investigated at 12 sites representing 5-10% of the natterjack toad localities presently known in Denmark. The expected heterozygosity (H _E) within each locality was generally low (range: 0.18-0.43). Further analyses failed to significantly correlate genetic diversity with population size, degree of isolation and increasing northern latitude, indicating a more complex combination of factors in determining the present genetic profile. Genetic differentiation was high (overall ¿ = 0.29) and analyses based on a Bayesian clustering method revealed that the dataset constituted 11 genetic clusters, defining nearly all sampling sites as distinct populations. Contemporary gene flow among populations was undetectable in nearly all cases, and the failure to detect a pattern of isolation by distance within major regions supported this apparent lack of a gene flow continuum. Indications of a genetic bottleneck were found in three populations. The analyses suggest that the remaining Bufo calamita populations in Denmark are genetically isolated, and represent independent units in a highly fragmented gene pool. Future conservation management of this species is discussed in light of these results.