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Not AvailableBoerhavia diffusa (B. diffusa), also known as Punarnava, is an indigenous plant and an important component in traditional Indian medicine. The accurate identification and collection of this medicinal herb is vital to enhance the drug’s efficacy and biosafety. In this study, DNA based molecular technique has been applied to identify and distinguish B. diffusa from its closely-related species B. errecta and their widely used adulterant Trianthema species. A protocol for extraction of total genomic DNA was optimized and found that 4% CTAB produced better quality and quantity of DNA for fresh leaves. A total 60 numbers of RAPD primers of OPN, OPF and OPQ series 1-20 were screened for the identification of polymorphic, reproducible and species specific markers among the three species. Among them 8 primers namely, OPN 5, 7, 8, 9, 10, 15, 16 and OPQ1 were found to produce polymorphic band in any of the three species. A total of six species specific bands (two of each species) were cloned and sequenced and from their nucleotide sequences, species specific SCAR markers developed. B. errecta specific SCAR marker, BEN-07- 1 and BEN-07-2 produced 408bp and 487bp amplicons exclusively in B. errecta whereas Trianthema specific marker TMM-01 produced 206bp amplicon in Trianthema and no amplicons were obtained in B. diffusa and B. errecta. The markers developed are efficient and reliable in authenticating the B. errecta, B. diffusa and its adulterant Trianthema. These markers could be used as molecular pharmacognostic tool in quality control of raw drug.Not Availabl