Amplification success and species discrimination efficiency of universal DNA barcode primers ( trnH - psbA , trnL , ycf1b , atpF-atpH , matK and rbcL ) was evaluated in 46 representative medicinal plant species of 28 families on agarose gel. The results showed that amplicons length polymorphism revealed by the primers atpF - atpH , trnH - psbA and trnL can simultaneously discriminate all the 46 species under study precisely. Some of the plant species included in this study are used as potential adulterants to other plant species. We were able to successfully discriminate the plant species from their potential substitute. Vitex negundo is an adulterant of Ocimum sanctum which has been successfully discriminated by all these three markers. Another example of adulteration between Bacopa monnieri and Centella asiatica was successfully discriminated by atpF-atpH , trnL and trnH-psbA on the basis of variability in amplicon length of these two medicinal herbs. Further, Cassia tora and Cassia fistula are also adulterants for each other and variability in amplicon length between these two species was revealed by atpF-atpH and trnH - psbA markers. A colour code distance matrix based on amplicon length polymorphism was designed to select primers which can effectively discriminate plants species on the basis of their amplicon length. Discrimination of plant species with the universal markers on agarose gel is a noble and inexpensive approach as it does not require sequencing of amplicons. This procedure will provide a way for the development of diagnostic markers to identify adulteration not only in herbal drug formulations but also in food material., Competing Interests: Compliance with ethical standardsOn behalf of all the authors, the corresponding author states that there is no conflict of interest.