Blueberry (Vaccinium spp.) is a member of Ericaceae and the recent important small fruit crop. This study was to construct a DNA marker database for blueberry varieties in Korea using simple sequence repeat (SSR) markers. A set of 49 SSR primer pairs was tested to select polymorphic SSR marker in 6 varieties. With 49 primer pairs, seventeen primer pairs showed polymorphism, reproducibility and band clearance. The genetic relationship among 34 varieties by using 17 SSR markers was analyzed. A total of 115 polymorphic amplified fragments were obtained by 17 SSR markers. Two to fifteen SSR alleles were detected for each locus with an average of 6.8 alleles per locus. Average polymorphism information content (PIC) was 0.671, ranging from 0.248 to 0.888. A total of 115 SSR marker loci were used to calculate Jaccard’s distance coefficients for cluster analysis by unweighted pair-group method with arithmetical average (UPGMA). Genetic distance ranged from 0.31 to 0.81 in 34 varieties and the dendrogram at a similarity 0.40 gave 3 main clusters according to blueberry species. Totally 34 varieties were identified by 17 SSR markers. Out of 17 SSR markers, a set of 3 minimum SSR markers was also enabled the identification of 34 varieties. Hence, we concluded that these SSR markers will be available for identifying blueberry varieties and alternative choice to distinctness, uniformity, stability (DUS) examination in blueberry along with valid phenotypic data.