The genus Diaporthe Fuckel consists of a large number of species recorded as pathogens, saprobes, and endophytes. While more than 1200 species of Diaporthe have been recorded to date, only pathogenic strains of Diaporthe were reported from Japan, notably affecting economically important plants. This study successfully elucidated the diversity of endophytic Diaporthe spp. from Prunus sensu lato (s.l.), commonly known as the Sakura tree, along with the addition of two novel species of Diaporthe to the Japanese mycobiota. In this study, a polyphasic approach was employed, using multi-locus analysis of 5 loci, including internal transcribed spacer (ITS) region, partial sequence of translation elongation factor 1–alpha (TEF), beta-tubulin (TUB), histone H3 (HIS) and calmodulin (CAL) genes. Additionally, morphological observation was also conducted by inducing sporulation of the isolates in artificial media. As a result, two novel species were introduced: Diaporthe endoprunicola A.H. Ujat & Y. Hattori and Diaporthe pseudoamygdali A.H. Ujat & Y. Hattori, along with confirmation of previously documented Diaporthe species in Prunus s.l.