Cosmopolitan in the western areas of Europe as well as on other continents, the Ilex genus is interesting for its genetic, phenotypic, and biogeographic variabilities. Its insular/local distribution, according to existing data on the periphery of the central and southern European areas, represents a suitable case study with reference to the adaptive plasticity or acclimatization of the Ilex aquifolium L. species to new climatic conditions. The aim of the present study was to analyze the genetic variability at the genome level in four insular populations of Ilex aquifolium L., i.e., in three spontaneous populations from Romania (RO), Serbia (SR), and Bulgaria (BG) and a cultivated population from Hungary (HU). According to the obtained results, the most genetically similar populations among the four considered in this study were those from SR and RO. Genetic variation overlapped genes that were generally associated with metabolic regulation/transport factors, water, and abiotic stress factors. The analysis of single-nucleotide polymorphisms (SNPs) at the levels of the chloroplast and mitochondrion, from the point of view of their distributions at the gene level, identified two clusters: one that includes the native populations (BG, SR, and RO) and a second one including the cultured population from HU.