In this study, we aimed to analyze the genetic relationship of 22 blueberry cultivars and establish a stable and reliable blueberry cultivar identification protocol, which would benefit blueberry germplasm evaluation, identification and management as well as novel variety breeding. We optimized the SSR-PCR reaction system, screened 15 SSR markers producing unambiguous, reproductive and abundant polymorphic bands. A dendrogram of 22 blueberry cultivars was constructed using these 15 SSR markers. It showed great consistency of the dendrogram and genetic background of the analyzed blueberry cultivars. SSR markers NA961 and NA1040 were chosen as core markers from the 15 SSR markers for blueberry cultivar identification, which together could completely distinguish the 22 cultivars. Reference markers and fingerprinting were made using these two core markers. A convenient and reliable blueberry cultivar identification protocol by SSR markers was successfully established