DNA regions (ITS, ITS2, matK, rbcL and trnH) have been examined for authentication and differentiation between E. peplus, E. hirta and E. tirucalli collected from Taif-KSA. The sequences of Euphorbia species were submitted in Genbank as new records within this research. ITS, ITS2 and rbcL provided good identification and clear resolution for the species, whereas, ITS2 and rbcL showed no or very little evolution. The highest sequence lengths were detected by matK and IT'S in E. hirta, E. tirucalli and then E. peplus. Our gathered results of Tajima relative evolutionary values revealed that ITS, matK and trnH showed rates of acceleration of evolution (P-values<0.05) within their sequences. The phylogenetic trees did not show any variability between the three Euphorbia species and the other retrieved GenBank accessions rejecting their endemism to flora of Saudi Arabia. The results support the hypothesis that E. peplus, E. hirta and E. tirucalli occur in the natural areas of Taif as naturalized species.