AimsMolecular epidemiological techniques, such as pulsed-field gel electrophoresis (PFGE), or multilocus sequence typing (MLST) have facilitated our understanding of the transmission routes of nosocomial infections by Pseudomonas aeruginosa. However, they are time consuming and technically demanding. To perform molecular epidemiological analysis in a standard microbiology laboratory, we aimed to develop a simpler and effective molecular epidemiological technique based on the open-reading frame (ORF) distribution patterns detected by PCR, which we call PCR-based ORF typing (POT). Methods and ResultsTen ORFs from genomic islets, five ORFs from genomic islands, and the metallo--lactamases (MBLs) bla(IMP) and bla(VIM) were selected by comparing the whole-genome sequences of different Ps.aeruginosa strains (PAO1, PA7, UCBPP-PA14 and LESB58). These 17 ORFs were detected, along with a Ps.aeruginosa marker, using 9-plex and 10-plex PCR systems. The genotypes in the POT were compared to those obtained by using PFGE and MLST. ConclusionsUsing the POT method, molecular epidemiological analyses of Ps.aeruginosa can be completed in 4h. Significance and Impact of the StudySince this method is very easy to perform, even in standard clinical laboratories, it could be a valuable tool for monitoring daily infection control measures.