The stems of Berberis aristata DC. (Berberidaceae) are used in the South Asian traditional medicine as a key ingredient in formulations for eye care, skin diseases, jaundice, rheumatism and also in diabetes. B. lycium Royle and B. asiatica Roxb. are traded as equivalents of B. aristata. Conventional macro-morphology and microscopic examination does not aid in critically distinguishing the three species. DNA markers were developed by amplifying and sequencing the complete internal transcribed spacer region (ITS1, 5.8S rRNA and ITS2) from the genomic DNA, using universal primers. The markers developed are efficient and reliable in authenticating B. aristata, B. asiatica and B. lycium. These are useful as molecular pharmacognostic tool in quality control of raw drugs.