The Persian fallow deer (Dama dama mesopotamica) formerly widespread in the Middle East was described scientifically at the end of the 19th century and considered extinct ever since. In 1956 it was rediscovered in south-western Iran. As a result, several countries have undertaken actions to reintroduce this subspecies in its native territory. In 2007 the Christian Oswald Foundation, in close cooperation with Iranian institutions, launched plans of in situ and ex situ breeding actions, with its centre in the German Von Opel Zoo and with cooperative Mediterranean partner countries as Israel, to support conservation efforts under scientific control. We performed genetic studies to study the suspected hybridization with European fallow deer (Dama dama dama) and a commitment to preserve pureblood populations. We used a set of microsatellite loci to examine genetic variation and recent hybridization with the European fallow deer. All microsatellite loci used were polymorphic, but some were monomorphic within subspecies. The allelic richness was similar in both subspecies but the ‘private allelic richness’ was reduced to a half in the Persian fallow deer, signalling allelic loss due to genetic drift and inbreeding. Moreover, we showed the presence of two discrete groups representing the two subspecies, with no signs of admixture or hybridization. Furthermore, Persian fallow deer studied here belong to two pre-defined genetics groups: the wild and the (more genetically impoverished) captive populations of Persian fallow deer. Finally, the Persian fallow deer deserves a high conservation priority, both in the Iranian stock and in the captive populations, so as to avoid hybridization.