The Urticaceae family, encompassing 53 genera and 2625 species, has a subcosmopolitan distribution. Forsskaolea L. is a small genus of seven species. It is represented in Saudi Arabia by two species, Forsskaolea tenacissima L. and Forsskaolea viridis Ehrenb. The current research elucidated the validity of DNA barcoding in species authentication and taxonomic identification of F. tenacissima, indigenous to the western region of Saudi Arabia. In this investigation, the internal transcribed spacer (ITS) and ribulose bisphosphate carboxylase, large subunit (rbcL) gene loci were designated as molecular markers to facilitate precise taxonomic documentation and the detection of novel F. tenacissima variants sourced from the western region of the Kingdom of Saudi Arabia. The Basic Local Alignment Search Tool (BLAST) was used for this process. The ITS and rbcL markers exhibited robust amplification of F. tenacissima, underscoring their utility as universal barcodes. The ITS region not only aided in confirming the identification of F. tenacissima at the generic and species levels but also demonstrated a superior capacity for distinguishing between closely related species or variants within the genus. The phylogenetic analysis of the individual datasets of the DNA sequences of rbcL and ITS addressed the monophyly of the species under investigation. In contrast, the combined phylogenetic analysis could not address the monophyly of Forsskaolea species in Saudi Arabia. The phylogenetic analysis of individual and combined datasets successfully placed F. tenacissima within the monophyletic tribe Forsskaolea. This tribal placement was the first time such information had been included in the flora of Saudi Arabia.