Asiatic Citrus Canker disease is induced by Xanthomonas citri pv. citri (Xcc) and threatens most of Citrus species and cultivars in many citrus-producing countries. Two pathogenic variants with different host range were described within Xcc. The pathotype Xcc-A infects a wide range of citrus and some related genera, has a worldwide distribution and is a permanent threat for citriculture. In contrast, Xcc-A* is naturally restricted to two Citrus species, limes (C. aurantifolia) and alemow (C. macrophylla) in limited areas. A rapid and reliable test using molecular methods is useful for accurate identification at pathotype level of this bacterium, classified as quarantine organism. Several PCR-based diagnostic tools have been developed for the identification of Xcc. In a preliminary study, we showed a lack of specificity for most of them when assayed on a large collection of Xcc strains and non-target strains. The aim of this study was to propose a new diagnostic tool for detection and identification of pathotypes of X. citri pv. citri. A multiplex quantitative real-time PCR assay (qPCR) was developed using hydrolysis probes targeting two markers of the bacterium. PCR primers targeting genes involved in pathogenicity and/or plant interactions were designed from the complete sequence of the Xcc-pathotype A strain IAPAR 306. Their specificity was assayed on a collection of Xcc-A strains (n=21), Xcc-A* (n=14) and other genetically related Xanthomonas (n=23) (1). A specific Xcc sequence encoding a conserved hypothetical protein related to chemotaxism and a specific Xcc-A sequence in a housekeeping gene were retained for the qPCR assay. The specificity of the qPCR assay was verified on the same strains collection of Xcc strains and non-target Xanthomonas. The qPCR assay detected all the Xcc strains and identified the pathotypes from cultures and citrus plant samples artificially infected with the bacterium: a sample is tested positive for the presence of A strains if b