Objective: In this present study, the DNA barcoding method has been applied to define and authenticate collected plant material as W. somnifera from its closely linked species at the molecular level and the organization of trnH-psbA in W. somnifera and its potential as a DNA barcode. Method: The plant samples were collected from Pannapatti, Salem district. The tissue from the leaf was extracted. Isolation of the genomic DNA was carried out using CTAB technique. Polymerase chain reaction (PCR) was carried out and amplified a specific region of the chloroplast using trnH-psbA and analyzed PCR products by gel electrophoresis. The Basic Local Alignment Search Tool (BLAST) was used to identify sequences in databases. The sequence information was used to construct a phylogenetic tree by Maximum Likelihood Method using MEGA X. This tree-building tool is used to analyze the phylogenetic relationship of W. somnifera. Results: DNA yield was good with 50 ng. The purity of the DNA was also calculated and it was 1.7. Phylogenetic tree constructed showed maximum resolved topology for internal branches of 82% bootstrap value with species-specific clusters with W. somnifera. Conclusion: W. somnifera are an indigenous plant of India and an important plant in the Indian Traditional Medicinal System. To improve and enhance the utilization of this plant species for further study as medicine, accurate, proper identification and authentication is very important.