Castanea sativa is a valuable tree species in Hyrcanian forests, an evolutionary relict ecosystem whose communities suffer from overexploitation and fungal diseases. In the current study, three species delimitation methods were utilized with ITS regions sequencing to determine the taxonomic status of Septoria causing leaf blotch of C. sativa in Hyrcanian forests. The results indicated that the length of ITS region in the genus Septoria(extracted from Gen Bank) varied from 650 to 680 bp.There were almost three times more variable sites in ITS1 than in ITS2. The ITS2 secondary structure of Hyrcanian Septoria community had the highest similarity with Septoria castaneicola, except for some differences in helix II and III. Also, Hyrcanian samples had minimum genetic distances with S. castaneicola and maximum with Septoria quercicola. The maximum parsimony method divided the studied Septoria genus into three distinct clades, mostly located in clade I. Clade II consisted entirely of Septoria aciculosa, while clade III contained S. castaneicola as well as Hyrcanian samples. In the current study, three species delimitation methods were utilized with ITS region sequencing to determine the taxonomic status of Septoria cause leaf blotch of C. sativa in Hyrcanian forests. The results indicated that the length of ITS region in the genus Septoria (extracted from Gen Bank) varied from 650 to 680 bp. There were almost three times more variable sites in ITS1 than in ITS2. The ITS2 secondary structure of Hyrcanian Septoria community had the highest similarity with Septoria castaneicola, except for some differences in helix II and III. Also, Hyrcanian samples had minimum genetic distances with S. castaneicola and maximum with Septoria quercicola. The maximum parsimony method divided the studied Septoria genus into three distinct clades, mostly located in clade I. Clade II consisted entirely of Septoria aciculosa, while clade III contained S. castaneicola as well as Hyrcanian samples.