ObjectiveThe objective of this study was to investigate the antibiotic susceptibility, virulence factors and clonal relationship among Pseudomonas aeruginosa isolated from environmental sources, hospitalized patients and the surfaces of cockroaches in the ICUs of four hospitals in Hamadan, west of Iran. A total of 237, 286 and 156 bacterial isolates were collected from clinical, environmental and cockroach sources respectively from May to September, 2017. The antimicrobial susceptibility was determined using disk diffusion method. The virulence factors, exotoxins A, S and U were detected by PCR. The genetic linkage of P. aeruginosa isolates were analyzed by Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR.ResultsAccording to our findings, 58 (24.4%), 46 (16%) and 5 (3.25) P. aeruginosa were isolated from clinical, environmental and cockroach samples respectively. The MDR phenotypes were detected in 18 (45%) and 15 (37.5%) of clinical and environmental strains. The environmental isolates harbored more exoA and exoS than did clinical isolates. Genetic diversity was established among P. aeruginosa isolates as 14 different ERIC fingerprints were detected. The clonal relationships was detected among clinical, environmental and cockroach isolates. Our results highlighted the importance of identifying and controlling the potential sources of P. aeruginosa infections in hospitals.