In this study, we established the rapid quantitative detection of metallo-beta-lactamase-producing Pseudomonas aeruginosa in clinical isolates and samples using real-time polymerase chain reaction (PCR) targeting gyrB (identification of P. aeruginosa) and blaIMP. The relative sensitivities and specificities of this real-time PCR assay were as follows: 100.0% and 100.0% for clinical isolates, and 100.0% and 98.4% for clinical specimens, respectively. The relative sensitivities and specificities of blaIMP-PCR were 100.0% in both clinical isolates and clinical specimens. The present PCR assay was easily and quickly performed, and it accurately detected P. aeruginosa and metallo-beta-lactamase.