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Da-Lat is a hilly area located in southern Vietnam. Macrofungal diversity of Da-Lat was investigated from 2018 to 2019. A total of 468 macrofungal specimens was collected and identified using the modern species concept and taxonomic and phylogenetic analyses. Among them, internal transcribed spacer(ITS) region of 401 specimens were successfully sequenced and compared with those of related species retrieved from GenBank. In total, 180 specimens were identified at the species level. The sequenced specimens were classified into 2 phyla, 13 orders, 38 families, 93 genera, and 124 species. The remaining 221 specimens (175 species) did not match the species level. This study is the first well-documented taxonomic list of macrofungi collected from southern Vietnam.