Measuring biological diversity is a crucial but difficult undertaking, as exemplified in oaks where complex morphological, ecological, biogeographic and genetic differentiation patterns collide with traditional taxonomy that measures biodiversity in number of species (or higher taxa). In this pilot study, we generated High-Throughput Sequencing (HTS) amplicon data of the intergenic spacer of the 5S nuclear ribosomal DNA cistron (5S-IGS) in oaks, using six mock samples that differ in geographic origin, species composition, and pool complexity. The potential of the marker for automated geno-taxonomy applications was assessed using a reference dataset of 1770 5S-IGS cloned sequences, covering the entire taxonomic breadth and distribution range of western Eurasian Quercus, and applying similarity (BLAST) and evolutionary approaches (ML trees and EPA). Both methods performed equally well, with correct identification of species in sections Ilex and Cerris in the pure and mixed samples and main genotypes shared by species of sect. Quercus. Application of different cut-off thresholds revealed that medium-high abundance sequences (>10 or 25) suffice for a net species identification of samples containing one or few individuals. Lower thresholds identify phylogenetic correspondence with all target species in highly mixed samples (analogue to environmental bulk samples) and include rare variants pointing towards reticulation, incomplete lineage sorting, pseudogenic 5S units, and in-situ (natural) contamination. Our pipeline is highly promising for future assessments of intra-specific and inter-population diversity, and of the genetic resources of natural ecosystems, which are fundamental to empower fast and solid biodiversity conservation programs worldwide.