The rapid spreading of the disease during last few years highlighted the need of a quick, sensitive and reliable method for Pseudomonas syringae pv. actinidiae (Psa) detection, to find possible inoculum sources and limit the pathogen spreading. A PCR method, using new primers designed on the gene encoding a putative outer membrane protein P1, was developed to detect Psa in symptomatic and asymptomatic tissue; a nested-PCR was also applied. Bleeding sap samples, collected in early spring from orchards with symptomatic and asymptomatic trees, were used both for PCR assays and for pathogen isolation and identification. The PCR and nested PCR methods were able to detect Psa presence at very low concentration from plant and pollen extracts; RFLP analyses with BclI on PCR and nested PCR amplicons confirmed the assay specificity, while the digestion with BfmI and AluI allowed to discriminate Psa strains isolated before 2008 from those isolated after 2008. Furthermore, the PCR and nested PCR on crude bleeding sap samples detected the presence of the pathogen in 3 and 5 of the 15 assayed samples, respectively. Direct isolation from the same samples and bacterial identification confirmed the results of molecular analysis.