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Six polymorphic sequence-tagged microsatellite sites (STMSs) were used to characterize 65 accessions of old garden roses [OGRs (Rosa L. spp.)] from seven botanical sections and 13 horticultural groups. Aims of the study were to define the genetic profiles of accessions and to provide information useful for the classification and pedigree reconstruction of OGRs. In roses, a precise botanical classification is difficult due to repeated hybridization carried out in breeding; OGRs are classified in horticultural groups on the basis of their original parentage or of their morphological traits. A total of 82 alleles were detected at six loci. The number of alleles per locus ranged from six to 21, with an average of 13.7 alleles per locus. A dendrogram was constructed by cluster analysis, displaying the relative genetic similarities between species' accessions, hybrids, and cultivars. Cluster analysis grouped the genotypes into seven major clusters that were substantially consistent with their classification into botanical sections and horticultural groups. Several hypotheses of apportionment of accessions to horticultural groups were evaluated on the basis of the relative position in the dendrogram of the analyzed individuals. Results demonstrated that DNA analyses can contribute to drawing the botanic classification of rose accessions, improving the genetic knowledge on the background of modern rose, and providing the basis for breeding programs.