Medicinal plants are the prime source of herbal materials, with 1178 species traded in India and 242 having high trade volumes. Most of the medicinal plants are collected from the wild indiscriminately, as very few of these plants are cultivated, leading to overexploitation and decline of species. Scarcity of these often results in adulteration and substitution, making traditional identification difficult. DNA barcoding has proved to be an accurate and reliable alternative for identification of such herbals but before being used for this purpose, species-specific barcodes for the species of interest should be available. The barcode library developed was used for authenticating the botanical identities of 163 herbal samples, procured from different markets or online, supposed to be belonging to 54 species, including 41 species of high trade volume by phylogenetic tree (NJ) method and BLAST1 analysis. Herbal samples (147) of 54 species could be tested and 92 (62.6%) of the tested samples were found to be authentic by using any of the four barcode loci. Of these, number of samples identified by ITS2 (69) was the highest, followed by rbcL (51), matK (26) and ITS (17) individually. BLAST1 search of the sequences of the samples not found to be authentic revealed that some of the herbal samples were substituted following the ayurvedic principle “Abhava Pratinidhi Dravyas” with their known substitutes or other unrelated medicinal plants, while few by totally unrelated plant species, such as, ‘Besan’ (Cicer arietinum) in place of ‘Vachhnag’ (Aconitum ferox) and an obnoxious weed, Parthenium hysterophorus substituting for ‘Pashanbheda’ (Bergenia ligulata).