DNA barcoding is a molecular tool that uses short gene sequences taken from a standardized portion of the genome for species identification. It is entering a new phase of applications to address questions relating to taxonomy, ecology, evolution, and conservation of wildlife. Medicinal plants contain high value ingredients, which are being used in drug development pharmacopoeial, non- pharmacopoeial or synthetic drugs. In this study, 70 medicinal plants were collected from Mardi-Himal. Fresh leaves of economically important and endangered plants were collected and preserved in silica gel. Plant DNA was extracted using widely used CTAB protocol and Qiagen kits. Both chloroplast (rbcL) and nuclear markers (ITS) were used for PCR optimization. The amplification success rate for rbcL and ITS markers were found to be 87.2% (61) and 85.7% (60) respectively. High quality purified PCR products were used for sequencing. Sequence analysis of both markers have identified and confirmed taxonomic identities of 67.14 %(47) plant samples. The research highlights the use of molecular markers in species identification; identify adulteration, authentication of herbal products and sustainable utilization and conservation of medicinal plants in Nepal.