The karyotypes of 21 herbaceous peony (Paeonia) cultivars were studied using root tip squashes revealing a wide variety of ploidy levels. There were three tetraploid (2n = 4x = 20), 11 triploid (2n = 3x = 15), and one diploid (2n = 2x = 10) cultivars in the hybrid group; five triploid (2n = 3x = 15) cultivars in the Itoh group; and one diploid (2n = 2x = 10) cultivar in the lactiflora group (LG). The asymmetry index (AI) ranged from 59.61% (‘Cytherea’) to 64.03% (‘Little Red Gem’). The karyotypes of all peony cultivars were 2A with 60% metacentric (m), 20% submetacentric (sm), and 20% subtelocentric (st) chromosomes. The karyotypic background of these 21 herbaceous peony cultivars is discussed in the context of the evolution of ploidy and the three cultivar groups. These results provide cytological information that would assist in a peony hybridization program.