Since 2008, Pseudomonas syringae pv. actinidiae virulent strains (Psa-V) have quickly spread across the main areas of kiwifruit (Actinidia deliciosa and A. chinensis) cultivation causing sudden and re-emerging outbreaks of bacterial canker to both species. The disease caused by Psa-V strains is considered worldwide as pandemic. Recently, P. syringae strains (ex Psa-LV, now called PsD) phylogenetically related to Psa-V have been isolated from kiwifruit, but cause only minor damage (i.e. leaf spot) to the host. The different biological significance of these bacterial populations affecting kiwifruit highlights the importance of having a diagnostic method able to detect Psa-V, which is currently solely responsible for the severe damage to the kiwifruit industry. In order to improve the specific molecular detection of Psa-V, a real-time PCR assay has been developed based on EvaGreen chemistry, together with a novel qualitative PCR (PCR-C). Both methods are based on specific primer sets for the hrpW gene of Psa. The real-time PCR and PCR-C were highly specific, detecting down to 50 and 200 fg, respectively, and were applied to a range of organs/tissues of kiwifruit with and without symptoms. These methods are important tools for both sanitary and certification programmes, and will help to avoid the spread of Psa-V and to check possible inoculum sources. In addition to being used as routine tests, they will also enable the study of the biology of Psa-V and the disease that it causes, whilst avoiding the detection of other populations of related P. syringae present in kiwifruit.