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From the earliest, Erwinia amylovora, the causal agent of the fire blight disease was considered an invasive pest that threatened several pome fruit varieties elsewhere and disrupted their commercialization. In this work, highly specific sets of real-time PCR fluorogenic primers and probes flanking the 16S-23S rRNA intergenic transcribed spacer regions have been designed based on TaqMan chemistry. Throughout a serial of validation trials, crucial reaction parameters have been optimized. Therefore, the primer set ITSEA2 highlighted the most reliable signals in terms of gene expression. With respect to conventional PCR tools, the newly developed molecular markers provided more accurate specificity, a higher sensitivity up to 105 cfu/ml and a quantitative amplification of the targets.