BACKGROUND: The global prevalence of herbal-based health care rapidly promoted requirements for medicinal plant resources. Accurate classification and identification are crucial to assuring the safety of these herbal sources. RESULTS: Here, we took Dendrobium (Orchidaceae), a famous horticultural and medicinal plant taxon, as the study focus to establish an effective authentication approach for medicinal plants based on new mtDNA barcodes. We first de novo assembled three complete mitogenomes using Illumina and Nanopore data. These three mitogenomes were 635,454 bp-831,745 bp long with multichromosomal structures. Moreover, the three mitogenomes were compared to the other four published Dendrobium mitogenomes. The results revealed great variations of the structure and repeat contents among these mitogenomes, while gene contents and genomic sequences were relatively conserved. The analysis of mutational hotspots showed eight mitochondrial DNA regions with high sequence variability (>
5%) at the interspecific level, which could provide abundant informatic loci for phylogeny, genetic diversity, and identification analyses. We also newly obtained mitochondrial sequences of 45 individuals from 15 Dendrobium species for authentication analysis. These 15 Dendrobium species were successfully identified by the whole mitogenome sequences and the isoform combination (Mt17 + Mt19) respectively. CONCLUSIONS: Our findings revealed that mitochondrial isoforms (chromosomes) could be used as super-barcodes for Dendrobium species authentication. The multi-chromosomal structure of mitogenomes provided new insights into the accurate authentication of medical plants.