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The Green Toad (Bufotes viridis) in Germany is mostly confined to secondary habitats and is experiencing severe population declines especially at its northwestern range boundary in the city of Cologne. As a basis for conservation management of this species, viz. to investigate the status of the population structure, we used a library of over 15,000 short tandem repeats to establish a new set of 12 microsatellite markers for this species. We applied these markers to B. viridis individuals from six sampling sites in Cologne, and included outgroup samples from sites in northern and central Germany. The results suggest the sampled Cologne toads form four distinct populations, without an obvious distinction between sites east and west of the river Rhine. Only the green toads of three neighbouring sampling sites clustered as one population, supporting the initial hypothesis that individuals from proximate sampling sites are genetically more similar than those from more isolated sampling sites. However, mitochondrial DNA sequences revealed a haplotype east of the Rhine that was absent west of the river. Toads from the northernmost sampled population in Cologne Ginsterpfad were not genetically depauperate, indicating that the severe declines at this site did not lead to a loss of genetic variation and were not related to inbreeding. Some Ginsterpfad individuals were genetically highly distinct from others, which may be explained by human introduction. Despite the encountered population structure, the low number of private alleles in the populations west of the Rhine, and the uniformity in mitochondrial haplotypes with the exception of one deviant allele east of the Rhine, lead us to recommend a pragmatic approach for conservation management. We suggest to consider all Cologne Green toads as belonging to a single management unit, but propose that, wherever possible, populations east and west of the Rhine should not be mixed in reintroduction measures.