| Summary: | <i>Tuber aestivum</i>, one of the most sought out and marketed truffle species in the world, is morphologically similar to <i>Tuber mesentericum</i>, which is only locally appreciated in south Italy and north-east France. Because <i>T. aestivum</i> and <i>T. mesentericum</i> have very similar ascocarp features, and collection may occur in similar environments and periods, these two species are frequently mistaken for one another. In this study, 43 <i>T. aestivum</i> and <i>T. mesentericum</i> ascocarps were collected in Italy for morphological and molecular characterization. The morphological and aromatic characteristics of the fresh ascocarps were compared with their spore morphology. Afterwards, we amplified and sequenced the elongation factor 1-α (EF1α) locus and built maximum likelihood trees to assess phylogenetic similarities between the two species. <i>Tuber aestivum</i> and <i>T. mesentericum</i> sequences cluster into different clades, with <i>T. mesentericum</i> sequences divided into three different sub-clades. According to their morphological features, three samples (T7, T8 and T12) were classified as <i>T. mesentericum.</i> However, when fresh, these ascocarps lacked the typical phenolic aromatic note. These specimens fall into the sub-clade III of the <i>T. mesentericum</i> phylogeny, which has the lowest genetic distance from the <i>T. aestivum</i> clade.
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