The objective of the present study was to establish a system of real-time polymerase chain reactions (PCRs) for the specific detection of Yersinia pestis using the LightCycler™ (LC) instrument. Twenty-five strains of Y. pestis, 94 strains of other Yersinia species and 33 clinically relevant bacteria were investigated. Assays for the 16S rRNA gene target and the plasminogen activator gene (resides on the 9.5-kb plasmid) and for the Y. pestis murine toxin gene and the fraction 1 antigen gene (both on the 100-kb plasmid) were combined for the use in two multiplex assays including an internal amplification control detecting bacteriophage λ-DNA. Applying these multiplex assays, Y. pestis was selectively identified; other bacteria yielded no amplification products. The lower limit of detection was approximately 0.1 genome equivalent. Rat or flea DNA had no inhibitory effects on the detection of Y. pestis. The results obtained using the multiplex real-time assays showed 100% accuracy when compared with combinations of conventional PCR assays. We developed and evaluated a highly specific real-time PCR strategy for the detection of Y. pestis, obtaining results within 3 h including DNA preparation.