Background/Objectives: Modern sequencing technologies have transformed the identification of medicinal plant species and varieties, overcoming the limitations of traditional morphological and chemical approaches. This review explores the key DNA-based techniques, including molecular markers, DNA barcoding, and high-throughput sequencing, and their contributions to enhancing the accuracy and reliability of plant identification. Additionally, the integration of multi-omics approaches is examined to provide a comprehensive understanding of medicinal plant identity. Methods: The literature search for this review was conducted across databases such as Google Scholar, Web of Science, and PubMed, using keywords related to plant taxonomy, genomics, and biotechnology. Inclusion criteria focused on peer-reviewed studies closely related to plant identification methods and techniques that contribute significantly to the field. Results: The review highlights that while sequencing technologies offer substantial improvements, challenges such as high costs, technical expertise, and the lack of standardized protocols remain barriers to widespread adoption. Potential solutions, including AI-driven data analysis and portable sequencers, are discussed. Conclusions: This review provides a comprehensive overview of molecular techniques, their transformative impact, and future perspectives for more accurate and efficient medicinal plant identification.

Modern sequencing technologies have transformed the identification of medicinal plant species and varieties, overcoming the limitations of traditional approaches. To address the challenge of discriminating Toona sinensis varieties, we sequenced and compared 15 complete chloroplast genomes from five varieties in northern China. Although these genomes exhibited a highly conserved structure, we identified eight variety-specific simple sequence repeats (SSRs), two unique tandem repeats, and several hypervariable regions with elevated nucleotide diversity. Phylogenetic analysis demonstrated that whole chloroplast genomes provided the highest resolution for variety identification, outperforming conventional barcodes. Furthermore, we developed 13 specific primer pairs targeting variable regions, and PCR validation confirmed their reliable amplification across varieties. In addition, sequence-level validation by Sanger sequencing of representative SSR and tandem repeat markers revealed stable, variety-specific repeat copy number differences. These results demonstrate that the identified chloroplast markers can effectively discriminate closely related T. sinensis varieties. This study confirms that despite overall conservation, the T. sinensis plastome contains sufficient variation for reliable identification, providing a robust framework for future germplasm conservation and molecular breeding.