Hymenocrater comprises of 11 species throught the world. The genus is an Irano-Turanian elements, which has several usages in traditional medicine. Northeast of Iran is the main geographical region of Hymenocrater species. The aims of the peresent study was to delimit 11 species of Hymenocrater to determine the species relationships based on morphological and molecular data (cp-DNA sequence), to produce divergence time and to construct barcode gap using the internal transcribed sequence because of adulteration of this genus in local markets. According to the results, Neighbor-Net network and UPGMA dendrogram of morphological data can delimit Hymenocrater species. Meanwhile, analyses of Chloroplast sequences (psbA-trnH) revealed that the species relationship and delimitation are difficult and complicated due to gene flow, hybridization, gene duplication, reticulation and chloroplast capture. Divergence time of Hymenocrater species estimated from Bayesian Evolutionary Sampling Trees date were seven million years ago. The center origin of Hymenocrater in Iran is Razavi Khorasan. Genetic fingerprinting of correctly identified medicinal species provides standard for comparing plant specimens sold in the market with true to type medicinal plant. Therefore, ITS sequences are important for barcoding this medicinally significant plant. It is correctly identified medicinal species, which will be used s standard for evaluating market sold plants
