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Tricholoma matsutake is a rare, precious, and wild edible fungus that could not be cultivated artificially until now. This situation has given way to the introduction of fake T. matsutake commodities to the mushroom market. Among the methods used to detect food adulteration, amplification of species-specific diagnostic marker is particularly important and accurate. In this study, the Pol gene is reported as a species-specific diagnostic marker to identify three T. matsutake varieties and 10 other types of edible mushrooms through qualitative and quantitative PCR. The PCR results did not reveal variations in the amplified region, and the detection limits of qualitative and quantitative PCR were found to be 8 ng and 32 pg, respectively. Southern blot showed that the Pol gene exists as a single copy in the T. matsutake genome. The method that produced the purest DNA of T. matsutake in this study was also determined, and the high-concentration salt precipitation method was confirmed to be the most suitable among the methods tested. The assay proposed in this work is applicable not only to the detection of raw materials but also to the examination of processed products containing T. matsutake. [ABSTRACT FROM AUTHOR]