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Belonging to the genus Cenchrus with 16-22 species, Cenchrus ciliaris L. (syn. Pennisetum ciliare (L.) Link, buffelgrass) is a perennial, common in warmer regions of both hemispheres, growing as a C4 grass in a wide range of habitats. In the present study we determined chromosome number and nuclear DNA content (2C DNA) for 28 natural populations collected from northern to southern Tunisia. Three ploidy levels were found: one tetraploid population (2n = 4x = 36), three pentaploid (2n = 5x = 45), and 24 hexaploid populations (2n = 6x = 54). The hexaploid chromosome number has already been reported for Tunisian populations of C. ciliaris but tetraploid and pentaploid (2n = 45) are new for this area. The tetraploid population was found in the semi-arid north; pentaploids were mostly on the northern side of the arid region, while the hexaploids were located mainly in the arid southern Tunisian and Saharan region. 2C DNA values, assessed using flow cytometry, correlated with chromosome counts. Nuclear DNA content ranged from 2C = 3.03 to 4.61 pg, revealing three ploidy levels corresponding to 4x, 5x, 6x, and mean 2C DNA amounts were of 3.03, 3.7 and 4.48 pg, respectively. Each cytotype produced viable pollen. Flow cytometric seed screening neither proved nor disproved apomixis. The most frequent hexaploid populations seem best adapted to arid conditions in southern Tunisia. The monoploid value, 1Cx, was constant. The existence of pentaploid cytotype suggests hybridization ability between tetraploids and hexaploids. It appears that polyploidization is the major evolutionary mechanism in the speciation of C. ciliaris.