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Based on a multi-locus phylogeny of a combined dataset of ITS, LSU, tef1-α, and rpb2 and comprehensive morphological analyses, we describe three new species from the Melanosporum group of genus Tuber and synonymize T. pseudobrumale and T. melanoexcavatum. Phylogenetically, the three newly described species, T. yunnanense, T. melanoumbilicatum and T. microexcavatum, differ significantly in genetic distance from any previously known species. Morphologically, T. yunnanense is distinctly different from its closest phylogenetically related species, T. longispinosum, due to its long shuttle-shape spores (average the ratio of spore length to spore width for all spores (Qm) = 1.74). Tuber melanoumbilicatum differs from the other species in having a cavity and long shuttle-shaped spores (Qm = 1.65). Although T. microexcavatum sampled ascomata have relatively low maturity, they can be distinguished from its closely related species T. pseudobrumale by the ascomata size, surface warts, and spore number per asci; additionally, phylogenetic analysis supports it as a new species. In addition, molecular analysis from 22 newly collected specimens and Genebank data indicate that T. pseudobrumale and T. melanoexcavatum are clustered into a single well-supported clade (Bootstrap (BS) = 100, posterior probabilities (PP) = 1.0); and morphological characteristics do not differ. Therefore, based on the above evidence and publication dates, we conclude that T. melanoexcavatum is a synonym of T. pseudobrumale. By taking into account current knowledge and combining the molecular, multigene phylogenetic clade arrangement and morphological data, we propose that the Melanosporum group should be divided into four subgroups. Diagnostic morphological features and an identification key of all known species in the Melanosporum group are also included. Finally, we also provide some additions to the knowledge of the characterization of T. pseudobrumale, T. variabilisporum, and T. pseudohimalayense included in subgroup 1 of the Melanosporum group.