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Several species of Venturiaspp. cause scab disease on fruit trees: V. inaequalison apple, V. pirinaon pear, and V. nashicolaon Asian pear that is listed as a quarantine pathogen in several countries in the world. An emerging disease caused by V. asperataon apple has very recently been reported in France, Italy and China. Fruit tree scab causes high economic losses and requires frequent fungicide treatments in orchards. Early detection of these pathogens is important in the management of this disease and—in the case of V. nashicola—to prevent its introduction and spread in disease-free areas. Using genomic resources available on these species, we identified polymorphic regions between them to develop a set of real-time PCR assays enabling detection of the four species on symptomatic fruits and leaves. We focused in particular on V. nashicolato establish a comprehensive validation procedure. The assay proved to be effective for targeting this quarantine species, thereby ensuring the reliability of analysis results in the context of regulatory monitoring.