Genome duplication has played an important role in plant evolution. Variation in genome size within species is common, particularly in grasses, but rarely considered when planning restorations. We surveyed ploidy variation within one habitat (tall grass prairie) in one region (northeast Iowa, U.S.A.) to assess the risk of ploidy mismatch in restoration plantings. Genome sizes were estimated using flow cytometry for samples from 19 remnant prairies, 5 restoration plantings, and 2 seed sources. Intraspecific ploidy variation in remnant prairie populations was found for two species of grasses, Andropogon gerardii (big bluestem) and Panicum virgatum (switchgrass). Restoration seeds differed from remnants in ploidy for three species of grass, P. virgatum, Sorghastrum nutans (Indian grass), and Spartina pectinata (prairie cordgrass), and for one species of forb, Amorpha canescens (lead plant). In the case of S. pectinata, local ecotype seeds were found to consist of two different ploidy levels. Restorations in grasslands in the United States and elsewhere are likely to create mixed ploidy populations, probably resulting in lower reproductive success for the remnant population. Prevention of mixed ploidy populations will require the screening of restoration seed sources and regional surveys for ploidy variation.