Buffalograss [Buchloë dactyloides (Nutt.) Engelm.] is indigenous to the short-grass prairies of North America and is a polyploid series of diploid, tetraploid, and hexaploid individuals. It has a base chromosome number of x = 10. The distribution pattern of these ploidy levels is not well-defined, especially in the southern Great Plains. We predicted the ploidy levels of 273 buffalograsses from the southern Great Plains of North America using flow cytometry to measure cellular DNA content. The buffalograss accessions were grouped into four distinct ploidy level groups. Very few diploid accessions were collected (2.6% of the collection), and all were found in northwest Texas and eastern New Mexico. Tetraploid accessions (23% of the collection) were found exclusively in the western regions of the southern Great Plains. Hexaploids were the most prevalent ploidy level, representing 73% of the collection and found throughout the collection area. Pentaploid accessions were also found in field sites (1.8% of the collection). No clear pattern of adaptation for ploidy levels is apparent from these data. In other collections, cold hardiness appears associated with higher ploidy levels, but this pattern is not apparent in the southern Great Plains.