The genus Euphorbia L. in Egypt is represented by 40 species, a subspecies, and 3 varieties. Those species are distributed in almost all phytogeographical regions in the study area. The genus is well known for its medicinal importance. Moreover, it is characterized by various and sometimes anomalous plant habits that make them interesting for taxonomists. In this study, six DNA markers: matK, rbcL, ETS, trnL intron, trnL spacer, and the entire ITS region (ITS1+5.8S+ITS2), as well as subunits ITS1 and ITS2 were evaluated singly and in combination to investigate their usage as potential DNA barcodes. The Maximum Likelihood (ML) and BLASTn analyses were conducted for 37 individuals representing 26 species of Egyptian Euphorbia. The BLASTn comparison of the newly generated DNA sequences of the Egyptian Euphorbia species showed that ITS, ITS1 and ITS2 subunits displayed high levels of species discrimination. On the other hand, the ML analysis of the DNA sequences of trnL intron yielded a better resolved phylogenetic tree, compared to the other regions. We also presented the phylogeny of Egyptian Euphorbia based on DNA sequences of other markers: matK, rbcL, trnL, and the entire ITS region, with additional sequences from GenBank. Our results have shown that E. dracunculoides, E. hyssopifolia, E. lasiocarpa and E. granulata are probably not monophyletic. This study along with the widest taxon coverage in Egypt, emphasizes the importance of using DNA markers for precise identification and phylogenetic placement of the genus Euphorbia in Egypt within the whole genus.