Acacia tortilis (Forsk.) Hayne ssp. raddiana (Savi) Brenan (Family Fabaceae; subfamily Mimosoideae) is a pioneer tree of dry areas. In spite of its several uses, there are no any published studies which deal with genetic diversity of Acacia tortilis (Forsk.) Hayne ssp. raddiana (Savi) Brenan in Tunisia. For this reason, here we study its genetic diversity with morphometric (only in Tunisian populations), chromosome counting, and cytometric (in Tunisian and Ziambabwean populations) approaches. Morphological field studies of four Tunisian populations (Bouhedma, Haddej, Hajeb and Gtar, respectively, designated as A, B, C, D) of A. tortilis (Forsk.) Hayne ssp. raddiana (Savi) Brenan using a general linear model (ANOVA) revealed statistically highly-significant differences between populations for all examined characters (P \ 0.0001). Significant differences were found also between genotypes for the majority of morphological traits (P \ 0.0001) except the width of 10 pods (laP), weight of 10 pods (Wt) and seed number of 10 pods (Sn) (P C 0.110). The variability in the tree, pod and seed characters can be exploited for agro-industrial purposes. The DNA amount and chromosome numbers of populations Umguza and Gwaii River from Ziambabwe and of Tunisian populations A, B, C and D were determined. Tunisian populations had 2C nuclear DNA contents of 2.95-3.03 pg, and were shown to be tetraploid (2n = 4x = 52), whilst the two Ziambabwean populations had 1.39-1.40 pg and were diploid (2n = 2x = 26). It is suggested that the Tunisian populations are paleotetraploids, adapted to the morearid local conditions.