Quercusilex, evaluated within evergreen oaks, has a wide geographic distribution in the Mediterranean basin. Hybridization and gene flow are effective and frequently observed mechanisms in Q. ilex. Additionally, weak reproductive barriers between closely related taxa in zones of geographical contact further increase genetic diversity and subsequent taxonomic problems. Two morphological types, known as rotundifolia and ilex, are defined based on the variations between Q. ilex populations appearing as a result of all these factors. However, it is still controversial whether morphological types: ilex and rotundifolia are subspecies of Q. ilex or two separate species. In this study, short DNA sequences that consist of matK gene-partial trnK gene intron of chloroplast DNA and ITS1-5.8S rRNA gene-ITS2 of nuclear DNA were used to overcome such difficulties and to reveal the variations between Q. ilex populations. All Q. ilex populations based on both barcoding regions were determined and examined using the Molecular Evolutionary Genetics Analysis (MEGA 11). The analysis such as base substitutions, variable and parsim-info sites, transitional and transversional base substitution ranges (%), and nucleotide frequencies (%) was performed and transitional substitutions according to the transversional substitutions for both barcoding regions were observed in the high-value. Furthermore, the sequences belonging to nuclear DNA in comparison to other barcoding regions exhibited higher variable and parsim-info sites. Finally, Maximum Parsimony (MP) dendrograms for both barcoding regions were drawn to evaluate the populations belonging to Q. ilex in terms of their variations, phylogenetic-evolutionary relationships, and taxonomic status. Although both barcoding regions support the separation of Q. ilex populations based on different morphological types, matK gene- partial trnK gene intron sequences exhibited clearer and more informative results than ITS1-5.8S rRNA gene-ITS2 sequences.