Thirty six isolates of Phoma tracheiphila from Italy, the causal agent of the mal secco disease on Citrus species, were characterised by different molecular tools in comparison with representative isolates of other phytopathogenic Phoma species. These included analysis of the distribution of RAPD and microsatellite markers and sequencing of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The results obtained with 12 RAPD primers (92 markers) and 7 microsatellite primers (56 markers) suggest that Italian isolates of P. Tracheiphila are genetically homogeneous, leading to identical patterns upon amplification with all the tested primers. Accordingly, ITSI-5.8S-ITS2 sequences were highly conserved (98–100% identity along a 544-characters alignment) among all the isolates of P. tracheiphila. A neighbor-joining analysis of ITS sequences of P. Tracheiphila in comparison with those of other Phoma species, as well as with alignable sequences from anamorphic and teleomorphic taxa retrieved in BLAST searches, revealed a close relationship between P. Tracheiphila and Leptosphaeria congesta . A pair of P. Tracheiphila -specific primers was designed on the consensus sequence (555 residues) obtained from the alignment of the newly generated P. Tracheiphila ITS sequences. A PCR-based specific assay coupled to electrophoretic separation of amplicons made it possible to detect P. Tracheiphila in naturally infected Citrus wood tissue collected from both symptomatic and symptomless plants. The limit of detection was 10 pg of genomic DNA and 5 fg of the ITS target sequence.