Global blueberry (Vaccinium spp.) growing has increased exponentially in the last two decades, fueled by a very dynamic offer of new cultivars. In this scenario, misclassification of genotypes is a matter of concern, despite that good nursery management practices are in place in most countries and new molecular fingerprinting tools have become available elsewhere. In this framework, here we report the use of three highly informative microsatellite (SSR) markers, selected after evaluating the performance of 12 SSRs on 30 commonly planted blueberry cultivars. We present here the validation of this set, comprising markers CA344, CA421 and NA1040 tested on a set of 136 cultivars and lines, all of which can be differentiated by these markers. A dendrogram built with the generated data set grouped the rabbiteye genotypes in a separate clade. The whole polymorphic information content of this set of SSRs was 0.878 and the observed and expected heterozygosity index averaged 0.890 and 0.886, respectively. Additionally, we detected 14 genotypes that showed minor allelic variants, exhibiting mostly single changes in their patterns with respect to the reference cultivar. Overall, the frequency of mutations was 0.6%, considering the 18 allelic variants identified in approximately 3000 analyzed samples. Marker CA421 exhibited the largest frequency of mutations, with 16 out of the 18 variants identified. Plants carrying these variants corresponded to the most planted cultivars introduced to Chile, which have been extensively micropropagated. The implications of these variations for the traceability of plants based on their fingerprinting are discussed.