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Characterization of plants with DNA markers is an ideal approach for identification of medicinal plant species as they provide objectively analyzable data for accurate and fast identification. There are many medicinal plants described in ancient literatures that have not been exploited for their medicinal values. A documented treasure of traditional knowledge of Kerala’s Ayurvedic and folklore practices is the ‘Hortus Malabaricus’. Description of various useful medicinal plants and their use as single drug can be seen in this ethnomedicinal compendium. The plants Justicia betonica Linn., Thottea siliquosa Lam., Memecylon edule Roxb., Pajanelia longifolia (Willd) K. Schum.) and Bauhinia accuminata Linn described in Hortus Malabaricus are considered to possess immense therapeutic potentials. As the Ayurveda industry as well as practitioners are facing severe scarcity of required medicinal plants, search for establishing the claimed pharmacologic values of hitherto unexplored plants is to be promoted. For this purpose, their proper identification and authentication is needed as a first step of scientific experiments. The molecular characterization including DNA isolation and sequencing provides fast and accurate identification of medicinal plant species. In this project, the molecular characterization of Justicia betonica, Thottea siliquosa, Memecylon edule, Pajanelia longifolia and Bauhinia accuminata, including DNA isolation and quantification, NCBI-BLAST analysis and DNA sequencing was done. The sequence analysis of Thottea siliquosa, Memecylon edule, Bauhinia accuminata and Justicia betonica by BLASTN (NCBI) is revealed 99 to 100 % nucleotide sequence homology with different database sequences enabling species level confirmation.