We report the discovery of Amanita sinocitrina, a species previously unrecorded in Japan. The specimens were identified using morphological characteristics and molecular phylogenetic analyses based on the internal transcribed spacer and large subunit regions of the fungal nuclear ribosomal RNA gene sequences. We also provided a detailed description of the morphology and anatomy of naturally established ectomycorrhizae of Japanese A. sinocitrina on the host Pinus densiflora. The ectomycorrhizae of A. sinocitrina had a thick mantle with a plectenchymatous structure with ring-like hyphal arrangement in the outer layer and pseudoparenchymatous structure with angular cells in the middle layer. The rhizomorphs showed intracellular crystals and enlarged core hyphae with partially or completely dissolved septa. These ectomycorrhizal morphological characteristics were reproduced by in vitro ectomycorrhizal synthesis using a culture of A. sinocitrina and axenic P. densiflora seedlings.