Below-ground ectomycorrhizal communities are often species-rich, and monitoring their dynamics is important to understand the conditions that promote truffle fructification. Characterization of the different ectomycorrhizas (ECM) at the species level can now be achieved by combining detailed morphological and anatomical descriptions with molecular approaches. Following this strategy, we have characterized ectomycorrhizal biodiversity in an artificial Tuber melanosporum plantation. Although the plantation was unproductive, T. melanosporum mycorrhizas were the most present and two Tuber-like mycorrhizal morphotypes, named ECMm1 and ECMm3, showing distinctive features were found. Internal transcribed spacer (ITS) phylogenetic analysis demonstrated that ECMm3 is related to the Tuber rufum/Tuber ferrugineum species complex, whereas ECMm1 shows the highest ITS similarity with Tuber scruposum and fungi-colonizing Epipactis roots. The results presented here provide more insights into genetic variability, mycorrhizal morphology, and below-ground distribution of fungi associated with artificial truffle plantations.