An epidemic of the disease chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis, induced a massive decline of populations of the common midwife toad (Alytes obstetricans) inhabiting the Peñalara Massif (Guadarrama National Park, Central Spain) in the years 1997–2001. The disease outbreak caused the disappearance of about 90 % of populations, leaving only eight remnant breeding populations. In response to the disease-induced population decline, a captive breeding program was started in 2008. Populations were kept separate to minimize possible outbreeding depression. Here, we examined indices of genetic diversity and population structure in these remnant populations to inform future reintroductions. Analysis of ten microsatellite loci showed strong genetic structure between breeding sites suggesting little genetic exchange and relatively low global genetic diversity. In accordance with the demographic bottleneck observed in the last years we found strong evidence for a reduction in genetic diversity. Our results suggest that the captive breeding program should mix animals from multiple sites from the Guadarrama Mountain Range, but avoid the genetically most divergent populations.