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Atriplex halimus L. (Chenopodiaceae) (Saltbush) is a perennial species used as a fodder shrub for livestock in arid and semi-arid areas, particularly in North Africa. The aim of this work was to determine whether differences in ploidy level and/or nuclear DNA content exist among populations from widely-separated sites in Tunisia. We determined nuclear DNA content and chromosome numbers for populations of A. halimus from seven different locations (Gabes, Medenine, Tataouine, Monastir, Tunis, Sidi Bouzid, Kairouan). The chromosome counts showed that all the Tunisian populations, plus a population from Eraclea (Italy), were tetraploid (2n = 4x = 36) whereas a population from Cala Tarida (Spain) was diploid (2n = 2x = 18). With respect to nuclear DNA, the 2C DNA content of population Cala Tarida was estimated to be 2.41 pg. There was no significant difference among the tetraploid populations (or among plants within populations), whose 2C DNA content ranged from 4.92 to 4.97 pg.