This paper describes a comparison study of test methods and supports the use of real-time polymerase chain reaction (PCR) for the detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers in routine testing. These 2 bacteria are quarantine organisms under European Union (EU) regulatory control and testing for (latent) infections of these bacteria in seed potatoes is mandatory. Real-time PCR tests were performed on 276 routine potato tuber samples, including samples infected with either C. michiganensis subsp. sepedonicus or R. solanacearum, and the performance of these real-time PCR tests was compared with that of immunofluorescence (IF). Real-time PCR tests, using different primer sets and extraction and PCR protocols, proved to be sensitive and specific for the detection of C. michiganensis subsp. sepedonicus and R. solanacearum in potato tubers in routine testing, and performed at least as well as IF. Real-time PCR is a good addition to the detection protocols as laid down in EU regulations (EU Council Directives 2006/56/EC and 2006/63/EC).