DNA barcoding is a novel method of species identification based on nucleotide diversity of conserved sequences. The establishment and refining of plant DNA barcoding systems are more challenging due to the high genetic diversity among different species. We, therefore, tested the potentiality of the ITS and rbcL markers for the identification of two medicinal species plants Solanum nigrum and Solanum villosum, which were collected from the Botanical garden of faculty of Science, Ain Shams Univ., Egypt. In this study, biological sequence homology and divergence of amplified sequences were studied using the Basic Local Alignment Tool (BLAST). Both DNA barcoding regions (ITS and rbcL) showed good universality amplification in the two species. The sequenced regions revealed conserved genome information for future identification of different medicinal plants belonging to these species. The amplified conserved barcodes revealed different levels of biological homology after sequence analysis. The results clearly showed that the use of these conserved DNA sequences as barcode primers would be an accurate way for species identification and discrimination. In this work, the ITS and rbcl markers were used to discriminate and confirm the identification of two medicinal plants, it was found that the viability and potentiality of ITS region in the identification process for the two plants used is more efficiency than rbcl, where rbcl confirm the identification of two plants at the generic level, while ITS at the species level. The findings of the study would be applicable in the medicinal industry to establish DNA based identification of different medicinal plant species to monitor adulteration.