Festuca ovina L. is a fine fescue that is used as a low-input turfgrass. The ploidy levels of F. ovina accessions held by the USDA National Plant Germplasm System (NPGS) are unknown, limiting the use of the germplasm in breeding programs. The objective of this study was to determine DNA content and estimate ploidy of these accessions. Among 127 accessions, we identified a wide range of ploidy levels from diploid to octoploid. We also found the accessions with higher ploidy levels usually had larger seed size. These results will be informative to plant breeders and researchers using germplasm from the F. ovina collection and point to challenges in maintaining polyploid, outcrossing germplasm seed stocks in common nurseries.