Buffel grass (Cenchrus ciliaris L. syn. Pennisetum ciliare (L.) Link) is an economically important warm-season perennial used primarily as a forage and range grass in many of the drier regions throughout the subtropics and tropics. Because buffel grass has excellent drought tolerance combined with desirable forage traits, it is an important forage and range grass in parts of Africa, including South Africa. It was introduced into many of the arid subtropical regions of the world, primarily Australia, North America, and South America, and today the species is grown on more than 50 million ha worldwide. Knowledge of the ploidy, meiotic behavior, and fertility of the plants used in hybridization programs became important because success depended upon using meiotically stable parental lines with compatible chromosome numbers. Little is known about the chromosome number of the buffel grass accessions in the USDA National Plant Germplasm System (NPGS) because the species has small chromosomes, that makes them difficult to count using traditional cytological methods. The DNA content of 568 accessions of buffel grass in the USDA National Plant Germplasm System was determined through flow cytometry to predict their ploidy levels. Based on DNA content, 308 accessions were determined as tetraploids with 36 chromosomes, 139 as pentaploids with 45 chromosomes, 20 as hexaploids with 54 chromosomes, two as septaploids with 63 chromosomes, and 99 as aneuploids. Chromosome counts of selected plants confirmed ploidy levels from DNA findings. Chromosome number of euploid plants could be predicted from the DNA data. The findings from this investigation reveal that the accessions in the buffel grass collection consist of a wide range of cytotypes, including polyploids and aneuploids with varying chromosome numbers. This demonstrates the amount of chromosomal diversity that occurs within the species and this collection. Grass breeders who use the buffel grass germplasm in the NPGS in their breeding programs should ensure that they know the chromosome number of the material requested. The predicted ploidy levels in Supplementary Table 1 will provide that information to the breeders.