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<i>Quercus robur</i> and <i>Q. petraea</i> are, in addition to <i>Fagus sylvatica</i>, the main economically used deciduous tree species in Europe. Identification of these two species is crucial because they differ in their ecological demands. Because of a changing climate, foresters must know more than ever which species will perform better under given environmental conditions. The search for differentiating molecular markers between these two species has already lasted for decades. Until now, differentiation has only been possible in approaches with a combination of several molecular markers and a subsequent statistical analysis to calculate the probability of being one or the other species. Here, we used MiSeq Illumina data from pools of <i>Q. robur</i> and <i>Q. petraea</i> specimens and identified nuclear SNPs and small InDels versus the <i>Q. robur</i> reference genome. Selected sequence variants with 100% allele frequency difference between the two pools were further validated in an extended set of <i>Q. robur</i> and <i>Q. petraea</i> specimens, and then the number of markers was deliberately reduced to the smallest possible set for species differentiation. A combination of six markers from four nuclear regions is enough to identify <i>Q. robur</i>, <i>Q. petraea</i> or hybrids between these two species quite well and represents a marker set that is cost-efficient and useable in every laboratory.