Tuber aestivum and Tuber uncinatum share a complicated common history from a taxonomic point of view. Indeed, different previous studies have shown contradictory conclusions. For some authors, Tuber aestivum and Tuber uncinatum are one species and for others, they are two different species. In those studies, few phylogenetic markers have been used, mainly the ITS marker. However, it has become the standard in phylogenetic studies to combine several genes to obtain a more robust phylogenetic representation. In our study, we used 9 molecular markers and sampled fruiting bodies from different European populations including 14 Tuber “aestivum” types and 14 “uncinatum” types. The results show some genetic differences between samples for some of the analyzed sequences. However, no genetic differentiation that would support the separation of two different species was observed.