Despite its current status as one of the most notorious invasive species worldwide, Arundo donax (Poaceae) has been utilized extensively by humans across Eurasia for thousands of years. This study utilized DNA barcoding of two chloroplast genes (matK and ITS) to establish a scientific foundation for identifying Arundo species. Genomic DNA extraction was performed, followed by PCR amplification using designated primers and subsequent sequencing via Sanger sequencing. Comparative analysis of nucleotides demonstrated that the obtained sequences were suitable for analyzing phylogenetic relationships. It was demonstrated that Arundo donax had been accurately identified and showed close relatedness to other specimens of the same species within the identical cluster when using the ITS primer. Chemotypic identification of Arundo donax leaf extract by GC-MS analysis found that a total of 16 compounds were detected, among which Z-10-Pentadecen-1-ol (C15H30O) was screened as a characteristic compound. These findings underscore the efficacy of DNA barcoding for precise and consistent identification of A. donax. Moreover, investigating Arundo donax chemotypes carries substantial theoretical and practical implications for its utilization and classification in practice.