Background and Aims: This study examines the pattern of genetic variability and genetic relationships of wild olive (Olea europaea subsp. europaea var. sylvestris) populations in the north-western Mediterranean. Recent bottleneck events are also assessed and an investigation is made of the underlying population structure of the wild olive populations. <br/> Methods: The genetic variation within and between 11 wild olive populations (171 individuals) was analysed with eight microsatellite markers. Conventional and Bayesian-based analyses were applied to infer genetic structure and define the number of gene pools in wild olive populations. <br/> Key Results: Bayesian model-based clustering identified four gene pools, which was in overall concordance with the Factorial Correspondence Analysis and Fitch–Margoliash tree. Two gene pools were predominantly found in southern Spain and Italian islands, respectively, in samples gathered from undisturbed forests of the typical Mediterranean climate. The other two gene pools were mostly detected in the north-eastern regions of Spain and in continental Italy and belong to the transition region between the temperate and Mediterranean climate zones. <br/> Conclusions: On the basis of these results, it can be assumed that the population structure of wild olives from the north-western Mediterranean partially reflects the evolutionary history of these populations, although hybridization between true oleasters and cultivated varieties in areas of close contact between the two forms must be assumed as well. The study indicates a degree of admixture in all the populations, and suggests some caution regarding genetic differentiation at the population level, making it difficult to identify clear-cut genetic boundaries between candidate areas containing either genuinely wild or feral germplasm.