Chloroplast DNA (cpDNA) sequence variations are broadly used to investi-gate interspecific relationships among plants. The trnL-trnF spacer is located in the large single-copy region of the chloroplast genome. The objective of this study was to evaluate the level of genetic variability between the Algeri-an and Tunisian species Hedysarum pallidum (North African endemic spe-cies) and H. capitatum. The phylogenetic relationships among the studied species were investigated by means of maximum Likehood analysis of se-quence polymorphism from chloroplast trnL-trnF spacer DNA; the tree parti-tioned the studied species in two clades. In addition, some specific variations loci- were identified between the species. The observed variations suggest considerable differentiation among cpDNA which could be useful to identify species.