This study investigated the genetic diversity and population structure of a persimmon (Diospyros kakiThunb., 2n = 6x = 90) collection in South Korea by evaluating 9751 genome-wide single-nucleotide polymorphisms (SNPs) detected using genotyping-by-sequencing in 93 cultivars. The results of neighbor-joining clustering, principal component analysis, and STRUCTURE analysis based on SNPs indicated clear separation between cultivar groups (pollination-constant nonastringent (PCNA, 40 cultivars), pollination-constant astringent (PCA, 19), pollination-variant nonastringent (PVNA, 23), and the pollination-variant astringent type (PVA, 9)) based on the astringency types, while separation between PVA and PVNA-type cultivars was unclear. Population genetic diversity based on SNPs showed that the proportions of polymorphic SNPs within each group ranged from 99.01% (PVNA) to 94.08% (PVA), and the PVNA group exhibited the highest genetic diversity (He = 3.86 and uHe = 0.397).F(fixation index) values were low ranging from −0.024 (PVA) to 0.176 (PCA) with an average of 0.089, indicating a deficiency of heterozygosity. Analysis of molecular variance (AMOVA) andFst among cultivar groups indicated that variation within individuals was higher than that among the groups. PairwiseFst values among the groups ranged from 0.01566 (between PVA and PVNA) to 0.09416 (between PCA and PCNA), indicating a low level of cultivar type differentiation. These findings highlight the potential application of biallelic SNPs in population genetics studies of allopolyploids species and provide valuable insights that may have significant implications for breeding and cultivar identification in persimmon.