Chile, the major exporter of blueberries in South America, grows two species commercially, the highbush blueberry (Vaccinium corymbosum L.) and the rabbiteye blueberry (Vaccinium ashei Reade). Considering the increasing demands for this fruit, it is necessary to have reliable methods for genotyping and genetic traceability of the commercially grown cultivars. In this study, an inter-simple sequence repeat (ISSR) marker-based system was established to perform the genetic identification of these cultivars. Ten cultivars of V. corymbosum: ‘Bluecrop’, ‘Bluegold’, ‘Duke’, ‘Elliott’, ‘Legacy’, ‘Misty’, ‘Nelson’, ‘O'Neal’, ‘Sierra’ and ‘Toro’ and three of V. ashei: ‘Climax’, ‘Premier’ and ‘Tifblue’ were analyzed. The sensitivity and reliability of this molecular marker system was compared with identification by simple sequence repeats (SSR). Six ISSR primers were used and high levels of polymorphism among the cultivars (80% of polymorphic loci) were detected, with high repeatability. Using individual primers, distinguishing among cultivars was possible in three cases. However, using pairs of ISSR primers provided greater reliability in cultivar identification. This ISSR-based technology is a simpler, faster, and less expensive alternative to SSRs for genotyping blueberry cultivars and can be used in genetic traceability studies as well as genetic improvement programs.