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Truffles ( Tuber  spp.) are well-known as edible ectomycorrhizal mushrooms, and some species are one of the most expensive foods in the world. During the fruiting process, truffles produce hypogeous ascocarps; a trained pig or dog is needed to locate the ascocarps under the ground. Truffles in northern Thailand have been recorded in association with  Betula   alnoides  and  Carpinus poilanei . In this study, we investigated the soil mycobiota diversity of soil samples from both of these truffle host plants in native forests using environmental DNA metabarcoding to target the internal transcribed spacer 1 (ITS1) region of the rDNA gene for the purposes of investigation of truffle diversity and locating truffles during the non-fruiting phase. In this study, a total of 38 soil samples were collected from different locations. Of these, truffles had been found at three of these locations. Subsequently, a total of 1341 putative taxonomic units (OTUs) were obtained. The overall fungal community was dominated by phylum-level sequences assigned to Ascomycota (57.63%), Basidiomycota (37.26%), Blastocladiomycota (0.007%), Chytridiomycota (0.21%), Glomeromycota (0.01%), Kickxellomycota (0.01%), Mortierellomycota (2.08%), Mucoromycota (0.24%), Rozellomycota (0.01%), Zoopagomycota (0.003%), and unidentified (2.54%). The results revealed that six OTUs were determined to be representative and belonged to the genus  Tuber . OTU162, OTU187, OTU447, and OTU530 belonged to  T .  thailandicum ,  T .  lannaense ,  T. bomiense , and  T .  magnatum , whereas OTU105 and OTU720 were acknowledged as unrecognized  Tuber  species. From 38 locations, OTUs of truffles were found in 33 locations (including three previously known truffle locations). Thus, 30 collection sites were considered new locations for  T .  thailandicum ,  T. bomiense , and other unrecognized  Tuber  species. Interestingly, at 16 new locations, mature ascocarps of truffles that were undergoing the fruiting phase were located underground. All 16 truffle samples were identified as  T. thailandicum  based on morphological characteristics and molecular phylogenetic analysis. However, ascocarps of other truffle species were not found at the new OTUs representative locations. The knowledge gained from this study can be used to lead researchers to a better understanding of the occurrence of truffles using soil mycobiota diversity investigation. The outcomes of this study will be particularly beneficial with respect to the search and hunt for truffles without the need for trained animals. In addition, the findings of this study will be useful for the management and conservation of truffle habitats in northern Thailand.

Soil Metabarcoding Offers a New Tool for the Investigation and Hunting of Truffles in Northern Thailand