We mapped the coding single nucleotide polymorphisms in four toxin genes-exoS, exoT, exoU, and exoY-of the Pseudomonas aeruginosa type III secretion system among several clinical isolates. We then used this information to design a multiplex PCR assay based on the simultaneous amplification of fragments of these genes. Eight strains of known genotype were used to test our multiplex PCR method, which showed 100% sensitivity and specificity in this small sample size. This assay appears to be promising for the rapid and accurate genotyping of the presence of these genes in clinical strains of P. aeruginosa.