The diversity of stain fungi is important if wood is inhabited with various fungi, and the discoloration mechanism will be better understood. MiSeq amplicon high-throughput sequencing (Illumina®) is able to detect species richness (the number of species within a community) and species evenness (the sizes of species populations within a community). This study detected fungal diversity in discolored Mongolian pine for the first time by the MiSeq approach, focusing on the nuclear ribosomal internal transcribed spacer-1 (ITS1). The results show that the discolored wood was inhabited by a combination of microorganisms, more than 90% of which belong to Ascomycota fungi at the phylum level. The MiSeq method revealed not only all the inhabited fungal species but also their quantitative relation to each other. The dominant fungal species in sample A are Helotiales (34.1%) and Hypocreales (20.7%). The dominant fungal species in sample B is Nectriaceae (67.9%), while Hypocrea (34.7%) and Sporothrix (27.6%) are the dominant fungal species in sample C. It was confirmed via core microbiome analysis that the following fungi were common stain fungi in the three discolored samples: Fusarium spp., Aspergillus spp., Sporothrix spp., Penicillium spp., Trichoderma spp., Alternaria spp. and Cladophialophora spp. [ABSTRACT FROM AUTHOR]