Prunus avium cultivars widely used in northern Greece were investigated in terms of inter- and intra-cultivar genetic variation and DNA fingerprinting. Based on 11 simple sequence repeats loci, the average number of alleles per locus (Na = 2.82), probability of identity (PID = 0.327), polymorphic information content (0.451) and expected heterozygosity (He = 0.494) were within the range reported in similar studies. The most informative markers were BPPCT039 and EMPa018. The cultivars were clearly separated in both an unweighted pair group method with arithmetic mean dendrogram and a multivariate space ordination. Any two cultivars differed on the average at 6.30 loci. The null hypothesis of zero intra-cultivar variability was tested and could not be rejected. Two cultivars (Tragana Edessis and Tragana Sarakinon) were genetically similar, but not identical. This study, the first of its kind for sweet cherry in Greece, presents a useful molecular tool for resolving issues of intra-cultivar variability and synonimity and provides a warranty of genetic identity in the handling and management of local traditional germplasm.