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Background and aims: Hospital-acquired infections caused by multidrug-resistant Pseudomonas aeruginosa incline hospital stay and costs of treatment that resulted in an increased mortality rate. The frequency of P. aeruginosa high-risk clones producing carbapenemases was investigated in our clinical samples. Materials and Methods: In this cross-sectional study, 155 non-repetitive P. aeruginosa isolates were included from different medical centers of Iran. Antibiotic susceptibility testing was determined, and the presence of β-lactamases were sought by phenotypic and genotypic methods. The clonal relationship of all isolates was investigated, and multi-locus sequence typing (MLST) was used for finding the sequence types of carbapenemas-producers.Results: The most susceptibility rate was recorded for colistin (94.9%). MOX and FOX were found both as low as 1.95% (3/155). The most frequent ESBL was SHV with 7.7% (12/155) followed by PER, OXA-1, and TEM with the frequency of 7.1% (11/155), 3.2% (5/155), and 1.3% (2/155), respectively. Carbapenemases were detected in 28 isolates (18%). The most frequent carbapenemas was IMP with 9% (14/155) followed by NDM, 8.4% (13/155). OXA-48 and VIM were also detected both per one isolate (0.65%). MLST of carbapenem resistant P. aeruginosa isolates revealed that ST244, ST664, ST235, and ST357 were spread in subjected clinical settings. REP-PCR uncovered high genomic diversity in our clinical setting. Conclusion: Clonal proliferation of ST235 strain plays a key role in the propagation of MDR pattern in P. aeruginosa. Our data showed that high-risk clones has distributed in Iran, and programs are required to limit spreading of these clones.