Isolates of an unidentified Rhizoctonia sp. (UN isolates) were obtained from Japanese zoysia grass (Zoysia japonica Steud) that exhibited symptoms of a new sheath rot disease. UN isolates were binucleate and showed hyphal fusion with tester isolates of Rhizoctonia anastomosis group (AG)-D. Those isolates were compared with isolates of subgroups I and II of Rhizoctonia AG-D based on cultural morphology, hyphal growth rate at different temperatures, anastomosis frequency, pathogenicity, and sequence analysis of the internal transcribed spacer (ITS) region of ribosomal DNA genes (rDNA-ITS region). The mycelial color of UN isolates was light yellow which differs from AG-D I but is similar to AG-D II. Sclerotia of UN isolates were dark brown in color and larger in size (1 to 3 mm in diameter) than those of AG-D subgroup I (1 mm in diameter), whereas isolates of AG-D II produced white mycelial clamps 4 to 5 mm in size. Hyphal growth rate of UN isolates was slower than that of two AG-D subgroups at several temperatures, especially 25°C. In pathogenicity tests on Japanese zoysia grass, UN isolates showed moderate disease severity and lower pathogenicity than isolates of AG-D subgroups I and II. Sequences of the rDNA-ITS region within UN isolates were almost homologous, but had lower homology with subgroups AG-D I or II. Phylogenetic trees constructed using ITS sequences showed that UN isolates formed an individual cluster that differed from the clusters of the two subgroups. We propose that UN isolates are a new subgroup of Rhizoctonia AG-D, subgroup III, and the name of the disease is "spring-rot" on Japanese zoysia grass.