Although the genus concept of Phyllosticta s. str. (teleomorph: Guignardia) as defined by van der Aa is widely accepted, the species concept is still controversial because it is often based on the morphology on host plants. In this study, the culture characteristics within Phyllosticta s. str. were examined, and the phylogenetic relationships among Japanese species of Phyllosticta s. str. and its teleomorph Guignardia were analyzed using 18S rDNA sequences. Phyllosticta s. str. formed a monophyletic clade. ITS-28S rDNA sequences extracted from fungal cultures derived from various host plants were divided into two subgroups. The first group included cultures from a wide range of host plants and were mainly derived as endophytes from a symptom-less plant. In the second group, cultures from each host plant genus formed distinct clades; these were often isolated as leaf pathogens from diverse plants. Isolates belonging to the first lineage generally grew faster on oatmeal agar. To classify species of Phyllosticta it is necessary to consider an integrated approach such as molecular phylogeny, host plant, colony growth, symptoms, and morphological characteristics of the conidiomata.