Xanthomonas fragariae, the causal agent of angular leaf spot on strawberry, is a quarantine organism in strawberry propagation material in the European Union. For the reliable screening of planting material for latent infections, a real-time PCR assay based on Taqman (R) chemistry for the detection of X. fragariae was developed. Primers and probe sequences were based on a DNA fragment amplified by a previously reported X. fragariae-specific technique. The sequence of this genomic fragment had no significant similarity with any published GenBank sequence. Specificity of the designed assay was tested with an extended range of X. fragariae collection strains and isolates, with other Xanthomonas spp. and with unidentified bacterial isolates from strawberry plants. A nested PCR, which until now was the reference method for sensitive detection in planta, cross-reacted with the reference strain of Xanthomonas campestris pv. campestris. In combination with an elaborated DNA extraction procedure, the Taqman (R) PCR enabled reliable detection down to 300 colony forming units in a 100 mg strawberry leaf sample. The assay offers a new tool for epidemiological research and for sanitary control of plant material with low level or latent infections of X. fragariae.