Sweet cherry (Prunus avium (L.) L.) is one of the most important fruit crops of temperate climates. In Chile, the actual planted area is over 42 000 ha that produce over 260 000 t yearly. The accurate identification of sweet cherry cultivars is key for processes involved both in breeding new cultivars and along the production chain. In this study, we performed the molecular characterization of 87 sweet cherry genotypes cultivated in Chile, using nine microsatellite markers originally described for both peaches and sweet cherries. The analysis showed that 69 of these genotypes corresponded to unique cultivars, each harboring a unique allelic pattern. They could be differentiated using only five markers (BPPCT-037, BPPCT-039, BPPCT-040, PMS-30 and UCD-CH18). The remaining 19 genotypes could correspond to misidentified, mutated or even synonyms of the studied genotypes, since they have allelic patterns identical to one or more of the 69 individualized genotypes. Between 3 and 8 alleles per marker were identified, with a mean of 6, while the expected heterozygosity over the nine polymorphic loci averaged 0.72, ranging from 0.59 in UDP96-001 to 0.78 in BPPCT-040. Phylogenetic and population structure analyses showed that most cultivars were grouped according to their country of origin or the breeding program from where they were released, being also coincident with their presumed pedigrees. These results are the basis for a fingerprinting protocol, based on microsatellite markers, for sweet cherry cultivars.