Bermudagrass (Cynodon spp.) is a forage and turf crop commonly used worldwide. The USDA bermudagrass germplasm set is composed of plant introductions (PI’s) collected around the world and contains different Cynodon species, primarily C. dactylon. The collection was screened in a replicated trial in Florida for forage yield, leaf width, nutritive value (NV), and Bermudagrass Stem Maggot (Atherigona reversura) (BSM), which is an invasive pest to the southeastern United States that damages bermudagrass fields. The goal of this research was to determine ploidy level and genome size in this USDA collection, and evaluate the influence of ploidy level in the estimation of genetic parameters for BSM, leaf width, dry matter yield, and NV traits. For chromosome counts using classical cytogenetics techniques, root tips and meristems were collected from a set of PI’s with known ploidy. The PI’s and cultivars with known chromosome counts were used as internal standards to run flow cytometry and estimate genome size of the PI’s with unknown ploidy. Ploidy level was determined for all accessions and were used to estimate genetic parameters of phenotypic traits. By providing information on ploidy levels and genetic parameters, this research will support breeding efforts and future selections for forage bermudagrass.