The genetic variability of five populations of Quercus petraea originating from the Transylvania and Maramureș regions of Romania was investigated in this study to provide insights into the species’ adaptability, population dynamics, and potential for preservation in the face of environmental challenges. To achieve this, sequence-related amplified polymorphism (SRAP) markers, in conjunction with a set of 18 primer combinations, were employed. The outcomes of the analysis revealed a range of polymorphisms spanning from 69.78% to 85.75%. Additionally, the assessment of genetic diversity using Shannon’s information index (I) yielded values ranging between 0.2887 and 0.3955, while Nei’s gene diversity (He) exhibited a spectrum from 0.1833 to 0.2582. The analysis of genetic variability, conducted via molecular variance (AMOVA), unveiled that 9% of the genetic variation was attributable to differences among the populations, while a substantial 91% resided within the populations. A further investigation of the population structure revealed that the construction of a UPGMA dendrogram based on Nei’s genetic distances elucidated the presence of two principal genetic clusters, a finding that was reinforced by a Principal Coordinate Analysis (PCoA). The genetic diversity revealed by Quercus petraea using SRAP molecular markers offers promising potential for upcoming breeding programs to identify optimal genitors, facilitating the development of well-adapted oak populations in the Transylvania and Maramureș regions.