Fungi display a rich variety of sexual reproduction systems and are therefore good models to investigate sex evolution. Moreover, understanding the investment in sexual reproduction of edible fungi is a critical challenge for improving their cultivation. The Perigord black truffle (Tuber melanosporum Vittad.) is a heterothallic ascomycete associated with trees through ectomycorrhizas and forming highly prized ascocarps. The aim of this study was to unravel the sexual reproduction strategy (hermaphroditic versus male/female specialization) of T. melanosporum in a truffle orchard by disentangling the contribution of female and male genotypes to the formation of ascocarps in a five-year investigation. Few genotypes were hermaphrodites, co-occurring with numerous genotypes behaving only as female or male, revealing trioecy. The genetic diversity of the male genotypes was higher than female diversity, suggesting for male elements a higher recruitment from ascospores. Most of the female and male genotypes were transitory (present only one year), whereas some genotypes persisted for several years: female-fertile genotypes as mycorrhizas, and male-fertile genotypes as soil free-living mycelium. Contrary to other ascomycetes, a high number of female-fertile only genotypes was found. We hypothesized that the mycorrhizal life style favours female-fertile strains and therefore that the life strategy influences fungal sexual strategy.