Thesium viride is used in the treatment of jaundice, hepatomegaly, splenomegaly and ulcers. This research aimed to establish some identification parameters such as pharmacognostic characters and DNA barcode of T. viride to aid in identification of the plant. Methods: The plant material was examined microscopically for some diagnostic structures and its physicochemical parameters such as moisture content, extractive values and ash values were also determined. DNA extraction from the plant material and amplification of the DNA using chloroplast barcode markers rbcL and matK were carried out. Microscopical examination of the aerial parts of T. viride revealed the presence of unicellular covering trichomes, straight walled polygonal epidermal, paracytic stomata, cork and sclerenchyma cells and a distinguishable cambium layer. The powdered plant showed moisture content (5.33%), total ash (10.33%), acid-insoluble ash (3.17%), water-soluble ash (4.93%), alcohol-soluble extractives (4.87%), and water-soluble extractives (5.93%). Polymerase Chain Reaction (PCR) amplification produced clear bands of approximately 512 bp and 817 bp, for rbcL and matK respectively. The amplified products were sequenced using the Sanger sequencing method and analyzed using BLAST against sequences deposited in the GenBank, and the submitted sequence was assigned accession numbers PZ051249 and PZ804900 for rbcL and matK respectively. The obtained sequences showed high similarity with related Thesium species, supporting the molecular identification of T. viride. The combined use of pharmacognostic evaluation and DNA barcoding provided useful identification of T. viride and established reference data for its authentication. These findings contribute to the standardization, quality control, and safe utilization of this medicinal plant.