DNA barcoding is a valuable tool for molecular identification of plant materials, which is primarily employed to guarantee the nature and therapeutic value of all available medicinal plants. Identification is crucial for the illegal medicinal plant trade, safe use, and preventing adulteration. In this study, DNA barcoding was used to evaluate and confirm the identification of some medicinal plants from the El-Omayed Biosphere Reserve (OBR). Two DNA regions, rbcL and ITS were selected due to their universality, ease of amplification and sequencing, and ability to identify taxa at the generic and species levels. The ITS region demonstrated a higher capacity for species discrimination power than the rbcL region. It assisted in identifying four of eight plants more precisely than rbcL. Together, ITS and rbcL could be used for plant species identification, conservation, and trade control of valuable plant resources.