The American persimmon (Diospyros virginiana) is a slow-growing, moderately sized tree fruit native to the forests of Kentucky. This tree fruit is in the early stages of commercial production with many cultivars selected from the wild. Small orchards of commercially available cultivars are planted in Kentucky. Persimmons are normally dioecious, and female trees require cross-pollination to produce fruit. There are two races of persimmon: the tetraploid (60-chromosome) race is centered in the southern Appalachian region, while the hexaploid (90-chromosome) race generally occupies a range north and west of the tetraploid range. These ranges overlap in Kentucky. Because the ranges overlap, cross-pollination may cause sexual incompatibility, resulting in pollination without fertilization, and therefore seedless fruits of poor quality. The objective of this study was to assess the ploidy level of commercially available American persimmon cultivars and native Kentucky persimmon populations. Leaf samples were collected from 45 cultivars and advanced selections, as well as 45 trees from native populations in Bullitt, Barren, and Franklin Counties. Flow cytometer analysis showed that only four of the selected cultivars were from the tetraploid race: Ennis Seedless, Weeping, Sugar Bear, and SFES; the remaining cultivars were from the hexaploid race. Both hexaploid and tetraploid American persimmon trees were identified in the populations sampled in the Bullitt County locations, but only tetraploid races were found in Franklin and Barren Counties. Because pollen from native trees could result in seedless fruit formation of poor quality when native seedlings are used as pollinizers in commercial production of American persimmon, ploidy level of seedlings needs to be considered.