Citrus crops are affected by many fungal diseases. Among them, Citrus Black Spot caused by the ascomycete Phyllosticta citricarpa is particularly economically damaging wherever it occurs. Many other species of Phyllosticta are described on Citrus, but only P. citricarpa is considered a quarantine pest on the European continent. In order to prevent the introduction of this species into Europe, it is essential to have a detection test which can reliably identify it, and not confuse it with other species present on citrus, notably P. paracitricarpa . The latter taxon has recently been described as very close to P. citricarpa , and most detection tests do not allow to distinguish the two species. In this work, we exploited the genomic data of 37 isolates of Phyllosticta spp. from citrus, firstly to assess their phylogenetic relationships, and secondly to search for genomic regions that allowed the definition of species-specific markers of P. citricarpa . Analysis of 51 concatenated genes separated P. citricarpa and P. paracitricarpa in two phylogenetic clades. A locus was selected to define a hydrolysis probe and primers combination that could be used in real-time PCR for the specific detection of the quarantine species, to the exclusion of all others present on Citrus. This test was then thoroughly validated on a set of strains covering a wide geographical diversity, and on numerous biological samples to demonstrate its reliability for regulatory control. The validation data highlighted the need to check the reliability of the test in advance, when a change of reagents was being considered.