The barcoding of medicinally important orchids of Bangladesh has been done so as to confirm the traditional taxonomic identification of the species using phylogenetic analysis and match with existing gene bank sequences. Presently, twenty two medicinally important orchid species used by tribal people were collected from different parts of Bangladesh. All the species were identified and classified following traditional taxonomic methods. DNA barcodes were extracted from dried leaf sample following standard DNA extraction methods, PCR and sequencing. The maturase-coding gene (matK) and the ribulose 1,5-bisphosphate carboxylase-coding gene (rbcL) regions were used as DNA barcodes for identification and phylogenetic analysis. A total of twenty one medicinal orchid species have been sequenced with the rbcL gene and sixteen species with the matK gene. Most of the sequenced genes (17 in rbcL and 8 in matK) are new addition to the gene bank. Where the new submissions matched entries in the gene bank, they showed very similar sequences indicating the proper identification with traditional taxonomic methods. Phylogenetic trees based on matK data distinguish every species from each other but rbcL showed less species discriminating power than matK. Phylogenetic trees constructed on combined data showed three different groups with evolutionary trends in both terrestrial and epiphytic orchids. Importantly, DNA barcoding genes ensure proper identification of the medicinally important orchid species, in the present study. This approach is ideal as a tool for the identification of critical medicinally important species which are the origin of crude drugs. The present study may prove useful for the establishment of Intellectual Property Rights (IPR) for future drug discovery.