A number of transmissible Pseudomonas aeruginosa strains have been identified which potentially constitute an emerging threat to patients with cystic fibrosis (CF). We sought to identify DNA markers that were specific to a transmissible P. aeruginosa CF clone and evaluate these probes on a large collection of genotypically distinct P. aeruginosa strains. Using subtractive DNA hybridization, in combination with analysis using the P. aeruginosa PAO1 genome chip, DNA markers specific for or absent from the Manchester transmissible CF strain (MA) were identified. Five subtractive DNA hybridization markers (MA15, MA18, MA21, MA22, and MA30) were found to be specific to strain MA and were located within a novel 13,318-bp genomic island, designated the MA island. The MA island encoded 18 genes and consisted of two bacteriophage-like regions; one region encoded the MA-specific subtractive hybridization markers, while the other bacteriophage-like region contained a Vibrio cholera-like toxin gene. Probes MA15, MA18, MA21, MA22, and MA30 were all found to be specific to strain MA when a collection of 141 P. aeruginosa strains was examined by hybridization with each DNA marker. In contrast, a previously isolated DNA marker for the Liverpool transmissible CF strain, PS21, was not found to be specific, detecting two additional strain types in the collection screened. Both the Manchester and Liverpool strain types were not encountered in CF populations outside the United Kingdom. The MA genomic island and Vibrio cholera-like toxin gene within it constitute novel genetic factors associated with a transmissible P. aeruginosa strain and their role in pathogenesis remains to be determined.