Background : Gentinae Macrophyllae Radix is one of the traditional medicines originated from the roots of multiple plants, Gentiana macrophylla Pall., Gentiana straminea Maxim., Gentiana crassicaulis Duthie ex Burkill and Gentiana dahurica Fisch., in Gentianaceae. Multi-origin traditional medicine usually has adulteration problem based on the morphological similarity and/or misunderstanding the species. Therefore, accurate and reliable identification criteria to ensure drug safety and quality is necessary. Methods and Results : We collected four original species of Gentinae Macrophyllae Radix from plantations and markets in China and Korea. DNA barcoding with four barcoding markers (Internal Transcribed Spacer (ITS), rbcL, trnL intron, trnL-F intergenic sapcer) was performed. Intra-specific variation was observed in ITS nucleotide sequence however, successfully distinct four original species based on the nucleotide discrepancy while trnL intron has no difference. trnL-F intergenic spacer has two transitions(T→C and A→G) sites only in G. crassicaulis and rbcL shows one transition(C→T) site in G. dahurica and G. macrophylla. Phylogenetic relationship analysis of the Gentinae Macrophyllae Radix revealed two major clades – clade I including three groups, G. macrophylla, G. straminea and G. dahurica, and clade II including G. crassicaulis. This aspects was shown more clear with multi-region combined analysis. Conclusion : DNA barcoding will be accurate and powerful criteria for the analysis the origin of Gentinae Macrophyllae Radix. However, single region analysis might be deficient such as trnL intron, rbcL and trnL-F intergenic spacer results in this research. Multi-region combined analysis based on the multiregional DNA barcode markers will be overcome the disadvantage and also increase the precision.