Understanding intraspecific genetic variation is one of the principal requirements for the evaluation of tree species capacity to cope with intensive climatic changes, as well as designing long-term conservation programs. Herein, we evaluated the genetic diversity and genetic structure of seven pedunculate oak (Quercus robur L.) populations, located at the southern margin of its distribution range on the Balkan Peninsula (Serbia). The objective of the study was to propose future in situ conservation measures aimed at protection of pedunculate oak adaptive and neutral genetic diversity at the species rear-edge. Genetic diversity and structure were estimated using twelve highly polymorphic simple sequence repeat (SSR) markers. The mean expected heterozygosity (He) was 0.769, allelic richness (AR) 9.63, and private allelic richness (pAR) 0.79, indicating high genetic diversity in the studied populations. Genetic differentiation among the populations was low (Fst = 0.032). Structure analysis, the unweighted pair group method with arithmetic mean (UPGMA) showed the existence of two gene pools unrelated to the populations’ area of occurrence. Taking into consideration the results of the current study and previous conservation activities on the pedunculate oak in Serbia, as well as the importance of rear-edge populations in the long-term conservation of the species genetic diversity, we suggested establishing three additional gene conservation units for securing long-term sustainability of the species.