Knowledge of genetic identity, genetic relationships, ploidy level, and chromosome numbers can enhance the efficiency of ornamental plant breeding programs. In the present study, genome sizes, chromosome numbers, and genetic fingerprints were determined for a collection of 94 Ilex accessions, including 69 I. crenata. The genome size of the entire collection ranged from 1.50 ± 0.03 to 8.01 ± 0.18. Within the species of I. crenata, genome sizes varied (mean ± sd) between 1.98 ± 0.08 and 2.30 ± 0.04, with three outliers: 3.06 ± 0.04, 4.04 ± 0.09, and 4.19 ± 0.08. The chromosome counting results showed 2n = 40 for I. crenata accessions and confirmed the outliers as one triploid and two tetraploids. A high intra-specific genetic diversity in Ilex crenata was found, after genetic fingerprinting using genotyping-by-sequencing (GBS). The species I. crenata was separated into three clades, which coincided with intraspecific differences in genome sizes (mean ± sd) of 2.09 ± 0.006, 2.07 ± 0.05, and 2.19 ± 0.06 pg/2C per clade as mean values for the diploids. Applying a principal coordinate analysis (PCoA) to the genetic fingerprinting data of all species in the collection revealed a wide genetic variation, which has not yet been commercially exploited. These findings could form the basis for selectively breeding parents, in order to create more genetic diversity via intra- and interspecific crosses.