Prairie cordgrass (Spartina pectinata Link) has been reported as polyploidy having two cytotypes: tetraploid (2n = 4x = 40) and octaploid (2n = 8x = 80) but recently in Illinois one plant of a new hexaploid (2n = 6x = 60) cytotype was observed to naturally occurring in seed obtained from tetraploid plants. In order to determine the extent of this new cytotype, the 2C nuclear genome size of 147 individuals of prairie cordgrass, originating from natural populations in Illinois, were determined by flow cytometry using somatic G1 nuclei, and the results confirmed by chromosome counts. Based on the flow cytometric analysis, putative hexaploid and tetraploid plants were found, with their respective 2C genome size of 2.35 and 1.56 pg. Chromosome counts of 80 and 40 respectively confirmed the ploidy levels of these plants. This increase of polyploids induced a greater variability of morphological expression associated two ploidy levels. At the end of October, plants were harvested and measured on 19 morphological characteristics and variability between tetraploids and hexaploids was observed and appeared to be the genetic variable. Substantial differences in the time of flowering, the stomatal size, and tiller mass were observed between tetraploids and hexaploids. The presence of ploidy mixture in the prairie cordgrass offers unique opportunities for studying the formation and establishment of polyploidy under natural conditions, which could be related to the morphological variability, considered as an ultimate step in plant evolution.