Winterberry (Ilex verticillata) is a deciduous shrub within the Aquifoliaceae family that holds significant ornamental and medicinal value. However, the lack of systematic research on the genetic background and phylogenetic relationships among its cultivars has hindered germplasm conservation and breeding efforts. This study marks the first application of genotyping-by-sequencing (GBS) technology to analyze winterberry germplasm resources. Sequencing was performed on 79 samples from eight representative cultivars, and 3,411,968 high-quality single-nucleotide polymorphism (SNP) markers were developed using a de novo assembly strategy. Population structure analysis based on STRUCTURE indicated K = 8 as the statistically optimal number of genetic components according to the delta K statistic. However, when STRUCTURE results were interpreted together with principal component analysis (PCA) and phylogenetic reconstruction, the winterberry cultivars were consistently summarized into five major genetic clusters. Ilex verticillata ‘Winter Gold’ and I. verticillata ‘Winter Red’ shared highly consistent genetic backgrounds, indicating extremely close kinship; I. verticillata ‘Citronella’ and I. verticillata ‘Oosterwijk’ clustered closely together; I. verticillata ‘Red Sprite’ (Rizhao) and I. verticillata ‘Red Sprite’ (Dezhou), despite differing geographical origins, clustered together, demonstrating good genetic stability; and I. verticillata ‘Golden Verboom’ and I. verticillata ‘Little Goblin Red’ each formed independent genetic branches, possessing unique genetic backgrounds. This study concludes that GBS effectively reveals the complex genetic structure among winterberry cultivars. The findings not only provide accurate molecular evidence for cultivar identification and intellectual property protection but also lay a solid foundation for future hybrid breeding, including parent selection, identification of superior genes, and advancement of marker-assisted breeding.