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Termitomyces pakistanensis sp. nov. is a member of an edible genus generally distributed in Asia and Europe. This species has been described as new species based on its different morphology, and scanning electron microscopy (SEM) of basidiospores. The novelty and degree of endemism is confirmed by analyzing the genetic variation of the internal transcribed spacer regions (ITS1-5.8 S-ITS2) of the ribosomal DNA gene, a universal fungal marker. The evolutionary affinities of new species is also evaluated with Asian and European species by phylogenetic analysis based on ITS sequences. In our phylogenetic analysis, this genus is found monophyletic comprising of two monophyletic sub clades: Clade I, Microcarpus, with small sized fruiting bodies generally less than 5 cm without pseudorrhiza and Clade II, Macrocarpus, with large sized fruiting bodies generally more than 5 cm having pseudorrhiza. All collections of Pakistani species clustered independently in Microcarpus clade showing their endemic genetic makeup as it is clustering independently. A comprehensive description, photographs of the basidiocarps and Scanning electron microscopy (SEM) micrographs of spores are provided. RESEARCH HIGHLIGHTS: It has a new species from Pakistan to world based on the scanning electron microscopy and further confirmed by DNA barcoding. The exact shape and size of basidiospores of this novel species is first time introduced by using SEM analysis. This genus is rarely described from Pakistan. This paper has introduced a two clade, Microcarpus and macrocarpus, in the world for this genus., (© 2022 Wiley Periodicals LLC.)