Strain KNUF-20-NI020 was recovered from soil collected during a survey of soil fungi in Korea in Gunwi-eup, Gunwi-gun, Daegu. The strain formed pale luteous colonies with rust-colored reverses on potato dextrose agar, reaching a diameter of 25.2 ± 1.0 mm after incubation for 14 days. Mycelial growth was most pronounced at 25°C and was relatively higher at pH values ranging from 6 to 8. For molecular identification, multiple gene regions were analyzed. The internal transcribed spacer (ITS) region showed 99.8% sequence similarity to Keissleriella quadriseptata, and the large subunit ribosomal RNA (LSU), small subunit ribosomal RNA (SSU), and translation elongation factor 1-alpha (TEF1-α) gene sequences were also most similar to those of K. quadriseptata. Phylogenetic analysis based on concatenated ITS, LSU, SSU, and TEF1-α sequences placed the KNUF-20-NI020 strain in the same clade as K. quadriseptata, clearly separated from other related species. Based on the combined cultural characteristics and multilocus molecular phylogenetic analyses, the KNUF-20-NI020 strain was identified as K. quadriseptata. This study represents the first report of K. quadriseptata from Korea and the first record of the genus Keissleriella in Korea.