DNA barcoding is an efficient method for species level identification and also contribute in taxonomy and biodiversity. Bioinformatics plays very important role in analysis of barcode data. The cytochrome oxidase 1 (CO1) mitochondrial gene was successfully utilised for animal DNA barcoding but for plant it is difficult to standardise plant DNA barcode. Girnar Forest Sanctuary of Junagadh, Gujarat is very rich in medicinally important plants. Many researchers have identified many plant species of greater medicinal value by traditional method but in the present study the aspect was to study these plant species using molecular methods specially DNA barcoding. In the present study 6 different species which are very important in herbal medicine were identified using ribulose- 1, 5-bisphosphate carboxylase- (rbcLa) barcode. The species were identified efficiently by evolutionary relations with closely related species by constructing phylogenetic tress using neighbour joining method. Our study indicate that rbcLa was powerful barcode for species identification and also helpful in prevention of adulteration of medicinal plants.