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Quercus pubescens s.l. is a group of taxonomically intricate and highly debated deciduous white oaks widely distributed in southern Europe. The Apulia region occupies the south-easternmost part of the Italian Peninsula; the land-use pattern is based on extensive agricultural systems and only 10% is covered by forests that are mainly composed of oak woods. It is the region in Italy showing the highest number of oak species, among which four putative species of the Quercus pubescens group, have been reported in floras and checklists with uncertain taxonomic value because of the overlapping of diagnostic characters. In this paper, we carried out a molecular analysis on natural populations of Q. pubescens s.l. distributed throughout the Apulia region. Individuals from 24 pubescent oak populations were sampled and each tree was genotyped at 11 polymorphic microsatellite markers. Overall, the average expected heterozygosity (He) was 0.629, and the allelic richness (Ar) ranged between 2.130 and 7.187. No differentiation was observed among the populations investigated, and the genetic differentiation coefficient (FST) was 0.036. Gene flow among populations was found to be relatively high (Nm = 6.664). From a taxonomic point of view, the possibility of the coexistence of more than one species among the Apulian pubescent oaks reported in the taxonomic and syntaxonomic literature is not supported by the results of this molecular analysis.