Truffles are hypogeous ascomycetes which form ectomycorrhizal associations with the roots of various host plants, including species of Quercus, Corylus, Carpinus, Pinus, and Fagus. These symbiotic relationships facilitate the exchange of nutrients and enhance the fitness of both species. Several truffle species are economically relevant due to their distinctive aroma and culinary value; however, a decline in natural yields has been reported, driven by anthropogenic pressures and climate change. This decline highlights the need for a deeper understanding of truffle biology, particularly regarding the existing genetic variability to better understand population dynamics and adaptive responses to environmental conditions. Summer truffle (Tuber aestivum Vitt.) is widely distributed in Europe and is particularly abundant in Italy. In recent years, it has attracted increasing scientific interest owing to its ecological plasticity and the growing concern over the effects of climate change on its natural habitats. Rising temperatures and altered precipitation regimes, pose significant threats to natural truffle grounds, with potential consequences for fungal biodiversity and forest ecosystems. To elucidate the genomic basis of intraspecific variability and local adaptation in T. aestivum, 96 fruiting bodies were collected from ten geographically and ecologically distinct Italian regions (Apulia, Basilicata, Calabria, Campania, Emilia-Romagna, Lazio, Marche, Piedmont, Tuscany and Umbria). DNA was extracted from the gleba of each specimen, and shotgun sequencing was employed to obtain genomic data. The primary objective of this study is to evaluate genome-wide diversity across Tuber aestivum individuals and to identify genetic patterns underlying population differentiation and local adaptation. This comparative approach may uncover genomic determinants linked to ecological plasticity and adaptive capacity, offering valuable insights to inform strategies for the conservation of biodiversity and the sustainable management of natural truffle habitats in the context of climate change.