AimsThe purpose of this study is to identify species from genus Diaporthe associated with a medicinal plant Costus spiralis by ITS, EF 1-, TUB and CAL gens. Methods and ResultsThe 30 isolates from the genus Diaporthe associated with the medicinal plant Costus spiralis were characterized based on morphological characters and the microculture technique and grouped by DNA fingerprinting with the ISSP gene. Afterwards, a total of 12 isolates were selected for the identification of the species based on the comparative research on the blast through the sequences of the ITS gene. Phylogenetic Tree of Maximum Likelihood were generated with the ITS gene individually and with the genes ITS, TUB, CAL and EF1- combined with the Diaporthe species recognized and with the additional sequences obtained from GenBank for these species. ConclusionsIt was not possible to characterize the 30 isolates microscopically and macromorphologically through the microculture technique and the macromorphological characteristics. The 12 isolates selected based on the DNA fingerprinting profile identified phylogenetically, revealed five distinct species of Diaporthe which are present in C. spiralis. Significance and Impact of the StudyThe molecular analyses used in this study are excellent alternatives for species-level identification of Diaporthe associated with medicinal plants.