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The European shot-hole borer Anisandrus dispar (Fabricius) (Coleoptera Curculionidae Scolytinae) is a well-known ambrosia beetle living on shrubs and several ornamental and fruiting trees where it can cause heavy damages. Like other harmful xyleborine species, A. dispar can represent a potential threat outside its native region. Molecular diagnostic tools can lead to accurate identification of xylophagous species hidden in wooden matrix in phytosanitary surveys at entry points. A molecular assay based on qPCR TaqMan™ Probes was developed for the identification of A. dispar from different matrices. To setup and perform the test, DNA extraction was carried out from adults, larvae, and artificial samples of wood chips from oak healthy plants whose lysates were contaminated with a known amount of DNA of A. dispar adults. The assay has proven inclusive for A. dispar, and exclusive towards the non-target organisms, showing 100% analytical specificity. The limit of detection was 0.32 pg µL-1 for the samples of insect adult DNA of A. dispar, and at 0.8 pg µL-1 for the samples containing lysates of Quercus spp. and 0.1 ng µL-1 of A. dispar adult DNA. Repeatability and reproducibility showed low values independently from the matrix used for DNA extraction, confirming the possible use in diagnostics of biological samples even if not directly related to the presence of A. dispar developmental stages. The presented approach may be adjusted and applied for phytosanitary purposes to other quarantine pests and rapidly detect possible infestations in vegetal matrices globally traded. [ABSTRACT FROM AUTHOR]