Tuber is a rare edible and medicinal fungus with a global distribution. The Macrosporum lineage within Tuber is a relatively small and infrequent clade, which has been receiving increasing attention due to the presence of an endangered species, T. gigantosporum. However, the species diversity, phylogenetic relationships, geographic distribution, and ecological characteristics of the Macrosporum group remain poorly understood. Through extensive sampling and combining the accurate and available global specimen and mycorrhizal data, 19 Tuber specimens from Southwest China were studied based on morphology, molecular systematics, and ecology. The specimens were identified as T. calosporum, T. glabrum, and T. sinomacrosporum within the Macrosporum group, with detailed descriptions provided, particularly supplementing the taxonomic features of T. glabrum. A classification key for the Macrosporum group species is also provided. The geographic distribution of specimens and diverse hosts suggest that the Macrosporum clade displays wider ecological adaptability. This study is important to better understand the diversity and conservation measures of Tuber resources throughout the world.