Sweet cherries can be grafted onto a wide range of rootstocks belonging to the genus Prunus. The identification of sweet cherry rootstocks using morphological traits is almost impossible particularly during the dormant season, yet it is very important for the grower to know exactly the rootstock as this has a major influence on cultivar performance and agricultural practices. However, DNA-based molecular analysis carried out on actively growing shoot tips, leaves or dormant buds provides a good opportunity to reliably distinguish the rootstocks. Herein, we have integrated high-resolution melting (HRM) analysis, coupled with five simple sequence repeat (SSR) markers, in order to facilitate the identification of the most popular sweet cherry rootstocks in Greece. The five SSR loci used were highly informative and generated a unique melting curve profile of microsatellites for each of the six sweet cherry rootstocks tested. In particular, one marker, BPPCT002, with six HRM profiles was sufficient to discriminate all the sweet cherry rootstocks studied, highlighting its potential use for rootstock identification. Hence, this assay provided a flexible, cost-effective and closed-tube microsatellite genotyping method well suited to sweet cherry rootstock identification.