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This study reports PCR-based techniques for a reliable molecular identification of five species of white truffles: Tuber magnatum Pico, T. borchii Vittad., T. maculatum Vittad., T. dryophilum Berk. & Br. and T. puberulum Berk. & Br. The sequences of the ITS region of several Tuber spp. were analysed and a pair of primers was designed for each species under study. The selected pairs of specific primers can be used for simple, rapid, reliable and unambiguous identification during the three developmental phases of the truffle life cycle: fruitbody, mycelium and ectomycorrhiza.