Bacterial wilt of lucerne, caused by Clavibacter michiganensis subsp. insidiosus (Cmi) is an important disease of lucerne worldwide. Detection and identification of the pathogen, especially in symptomless plant material, is difficult and often requires plating on specific media. Three polymerase chain reaction (PCR)-based assays have been published for identification of Cmi. The present study reports on the results of experiments to evaluate these PCR assays and the development of a quantitative, real-time PCR assay. The assay is based on the intergenic spacer region flanked by 16 S and 23 S rRNA genes and is used for the detection of Cmi in DNA extracted from naturally infected lucerne plants. All assays were evaluated using strains of Cmi, closely related bacteria, 87 unidentified bacteria isolated from lucerne plants and DNA extracted from infected and uninfected lucerne plants. Results showed that the new assay is more specific and sensitive than previously published assays.