Hazelnut (Corylus avellana L.), a member of the family Betulaceae and the order Fagales, is native to Europe, Turkey, and the Caucasus. It has been cultivated for more than 5000 years. The majority of cultivars were selected from local wild populations over the past few centuries. Hazelnut is native to northern Portugal, with local production from landrace clonal selections. In recent years, hazelnut production has declined, and landraces are in danger of being lost. In this study, we used 16 microsatellite markers to investigate genetic diversity in wild populations, Portuguese landraces, and reference cultivars. A high level of polymorphism was found, as indicated by mean values for expected heterozygosity (0.74), observed heterozygosity (0.71), and polymorphism information content (0.78). A neighbor-joining dendrogram showed a clear separation of the wild genotypes from the landraces and reference cultivars. Bayesian analysis placed individuals in three groups, showing differentiation of wild populations, landraces, and reference cultivars. Our studies provide new insights on the origin of Portuguese landraces.