ABSTRACT Citrus bacterial canker disease has been introduced at least three times into Florida in the last 15 years and, despite federal and state quarantine and eradication efforts, continues to spread in Florida. Accurate, fast, and reliable detection of the causal agent is of great importance. However, citrus bacterial canker is caused by at least two groups of phylogenetically distinct Xanthomonas citri strains, and there is host range variation within both groups. We developed a fast, sensitive and reliable real-time polymerase chain reaction (PCR) assay using a portable, field-hardened RAPID machine and primers designed to detect all canker-causing strains. Single-lesion sampling methods were developed that required minimal handling and allowed complete real-time PCR diagnosis in a total time of 4 h and with an apparent sensitivity of less than 10 CFU of target cells from diseased lesions. This sensitivity allowed molecular detection for the first time of X. citri in a herbarium sample from a 1912 canker outbreak. Sensitivity was improved significantly by the use of CaCO(3) and Silwet L-77, and by either minimizing the amount of citrus lesion tissue sampled or by soaking or swiping but not grinding the lesions. Primer design also was of significant importance in both specificity and sensitivity.