AbstractDespite the high economic value of safflower (Carthamus tinctorius), the potential of several wild related species as genetic resources is still poorly studied, in particular with regards to drought tolerance, a priority in safflower improvement. This situation is partially explained by the numerous and still discussed uncertainties in phylogenetic relationships within the safflower species complex. In this study, we focused on two rare Algerian wild species, Carthamus strictus and Carthamus chouletteanus. We aimed at: (i) clarifying their taxonomic status, and determining their phylogenetic relationships with safflower and other Mediterranean species of Carthamus sensu lato (s.l.) (Carthamus sensu stricto (s.s.) and Carduncellus genera) by using nucleotide diversity within the internal transcribed spacer region (ITS) and (ii) assessing the water use efficiency (WUE) of these species by determining their carbon isotope composition. Our results support the existence of two well supported different clusters among the safflower species complex: Carthamus s.s. including Eastern annual species and the vast Carduncellus group constituted by Western perennial species, including the two studied Algerian species. Also, significant differences for carbon isotopic composition between Carthamus s.s. and Carduncellus were revealed. Carduncellus species displayed significantly higher δ13C values (i.e., higher WUE) than Carthamus s.s. species. Carthamus chouletteanus displayed the highest average value of δ13C (highest WUE) among the species studied. Our data encourage for further studies on the possibility of hybridisations between species of the two genera and suggest that Carduncellus should be considered as potentially more interesting genetic resources to improve the WUE of safflower, rather than Carthamus s.s. species.