Truffles are ectomycorrhizal fungi living in symbiosis with many trees and shrubs. Inoculated seedlings with truffles are available since forty years and the interest in truffle cultivation is increasing worldwide. However, novel management guidelines for sustainable truffle orchards, in which the production is less unpredictable even in the climate change context, are needed. A better understanding of the biological and ecological mechanisms driving the truffle life cycle is one of the approaches to reach these goals. The genomic resources available for Tuber melanosporum allowed us to unravel its sexual reproduction mode by identifying mating type genes and to characterize new polymorphic markers allowing population genetics analyses. The study of genet distribution in truffle orchards showed a non-random spatial distribution of ectomycorrhizas formed by both T. melanosporum mating types, as well as a rapid yearly turnover of these genets. This analysis revealed a striking competition between genets based on their mating-type genes to colonize plant root system and questioned the localization of both parents in the initiation of the sexual cycle. To gain additional information on T. melanosporum genetic diversity, we re-sequenced the nuclear genome of six geographic accessions. A total of 442,326 single nucleotide polymorphisms (SNPs) corresponding to 3,540 SNPs/Mbps were identified. Based on their SNP density, samples clustered according to their geographic origin and the putative role of the last glaciation in T. melanosporum phylogeography is confirmed. Additionally, genomic regions and genes potentially subjected to positive or purifying selection were identified. Finally, a large-scale analysis of T. melanosporum populations at European scale using highly polymorphic simple sequence repeats (SSR) is also in progress.