DNA barcoding has been proposed as a powerful taxonomic tool for species identification genetically. The Consortium for the Barcode of Life (CBOL) Plant Working Group has recommended the combination of rbcL & matK as the core plant barcode. Rhododendron of Nepal shows genetic diversity due to which 31 different species were reported till date, found in Nepal based on morphological character only. For the genetic identification first time, we collected different samples from the central and western parts of Nepal and also based on its phytochemical too. Cold extraction was performed using methanol as solvents. The crude extracts were tested for the presence of phytochemicals such as alkaloids, Saponin, glycosides, tannins, flavonoids, and Coumarins. The extract was also used for antimicrobial assay into five different pathogenic microorganisms. MIC was done for calculating the minimum effective concentration of the extract. Then TLC was done to separate the compound. The methanol extract was analyzed for core secondary metabolites by HPLC-QTOF-MS and found more than 20 important medicinal compounds. DPPH assay was also done with crude extracts for identifying antioxidant potency of the extracts. Among samples compared, Gulmi and Palpa showed the best result for antioxidant assay with minimum IC50. Similarly, phenolic content was also checked and samples from Palpa showed high phenolic content. Finally with the rbcL and MatK gene loci was amplified and sequenced and based on alignment with NCBI-BLAST databased it show the 99.98% similarly for the R.arboreum. The final contig was submitted to NCBI and get the accession number LC456605.1 and were submitted to the BOLD database for the final authentication and succeed for the first time to publish DNA barcode of Nepalese plants.