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The aim of this study was to develop a rapid, sensitive and reliable field-based assay for detection of the quarantine pathogen Pseudomonas syringae pv. actinidiae, the causal agent of the most destructive and economically important bacterial disease of kiwifruit. A comparative genomic approach was used on the publicly available P. syringae pv. actinidiae genomic data to select unique target regions for the development of two loop-mediated isothermal amplification (LAMP) assays able to detect P. syringae pv. actinidiae and to discriminate strains belonging to the highly virulent and globally spreading P. syringae pv. actinidiae biovar 3. Both LAMP assays showed specificity in accordance to their target and were able to detect reliably 125 CFU per reaction in less than 30 min. The developed assays were able to detect the presence of P. syringae pv. actinidiae in symptomatic as well as in asymptomatic naturally infected kiwifruit material, thus increasing the potential of the LAMP assays for phytosanitary purposes.