The genus Sphaerulina is plant pathogenic fungi that causes leaf spot diseases on various plants regardless of herbaceous or arboreous plants. The plant will exhibit symptoms identical to the leaf spot caused by Septoria spp. and are indistinguishable morphologically. Recently, the taxonomical re-examination based on the phylogenetic relationship and morphological characteristics was carried out worldwide. However, most Japanese Sphaerulina species are still recorded under the Septoria genus. This study aims to reveal the taxonomical position and diversity of Japanese Sphaerulina species based on the phylogenetic relationships and morphological characteristics and contribute to complementing the phylogenetic backbone tree for Sphaerulina spp. A total of 27 isolates deposited in the Culture Collection, Phytopathology lab., Mie University (MUCC), were analysed for those phylogenetic positions using a combined matrix composed of ITS, LSU, TUB, CAM, ACT, TEF, and RPB2 regions. Microscopic observation for morphological characteristics was also conducted on the herbarium specimens linked to the isolates examined in this study. As a result, the generated trees by RAxML and MrBayes software packages indicated the existence of eight new Sphaerulina species occurring on different host plants. Two of the species require transferring from the genus from Septoria to Sphaerulina. Moreover, these Sphaerulina species mainly inhabit arboreous plants and form each well-supported clade according to each host plant, as indicated in previous studies.