A set of 87 apple accessions, located in three Portuguese apple germplasm collections, plus eight reference cultivars, were analyzed using 16 SSRs with the aim of assessing their genetic diversity and structure and evaluating relationships among them. Among the accessions studied, 64 unique genotypes were identified, 51 diploids and 13 putative triploids, revealing 19 groups of synonyms and 4 of homonyms. The genetic analyses performed by Bayesian model-based clustering (Structure) revealed a clear differentiation of two major groups (RPP1 and RPP2), one of them (RPP1) corresponding to old local Portuguese accessions, some of them putatively derived from ancient hybridization with Reineta, and the second one (RPP2) including all reference accessions and local Portuguese accessions considered to be of more recent origin. These results were confirmed by factorial correspondence analysis (FCA) and molecular variance analysis (AMOVA) and are in accordance with the Jaccard coefficients. Results indicate that the Portuguese apple germplasm includes an important and distinct gene pool of cultivars with respect to reference international cultivars, being a relevant source of genetic diversity for present and future breeding programs, originated at least 200 years ago, when some of these cultivars were firstly named. Moreover, this study will be useful to improve the apple germplasm management and in the definition of a preservation strategy concerning the Portuguese apple landraces.