Background: Pseudomonas aeruginosa is an opportunistic bacterial pathogen responsible for a significant proportion of hospital infections, especially in intensive care units (ICUs). The bacterium's ability to resist multiple drugs and survive in hospitals makes it more likely to stick around and cause disease. In Diwaniyah Governorate, repeated studies have explored the phenotypic and molecular characteristics of Pseudomonas aeruginosa, particularly its beta-lactamase production and antibiotic resistance. The identification of genetic markers linked to drug resistance is essential for designing antimicrobial guided strategies. Even though previous studies have evaluated Pseudomonas aeruginosa in several hospital settings, little is known about its molecular characterization and resistance profile on this setting. Knowing the prevalence of resistant genes such as GES, blaOXA-48 and SPM may help in establishing measures for infection control. Objectives: The objective of this study was to use molecular techniques to isolate and identify Pseudomonas aeruginosa from clinical samples, find out what kind of antibiotics they are resistant to, and look for genes that make beta-lactamase. Materials: The study involved the different culture media were used, and identification relied on several biochemical reagents, such as Catalase, Oxidase, IMVC, and TSI tests, in addition to the Vitek2 Compact system. To detect β-lactamase activity, Penicillin G solution, starch solution, and iodine solution were applied in the rapid iodine method. For molecular work, genomic DNA was extracted using the Genomic DNA Mini Kit (Geneaid, USA). Preparation of electrophoresis gels required agarose powder (1.5%), TBE buffer (1X), and ethidium bromide as a DNA stain. The amplification of target genes was carried out using Wizpure PCR FDmix, along with specific primers for 16S rRNA, blaSPM, blaGES, and blaOXA-48 genes, in addition to PCR-grade water. Results: Pseudomonas aeruginosa was confirmed in 15 out of 40 clinical samples (37.5%). The highest isolation rate was found in burns (60%), then wounds (26.6%), and finally urine (13.4%). The rapid iodine test and the Vitek 2 system showed that 80% of the isolates made beta-lactamase, which means they were resistant to most antibiotics except for tigecycline. Polymerase chain reaction (PCR) analysis indicated that all isolates possessed the 16S rRNA gene, 75% contained the OXA-48 gene, 40% harboured the bla GES gene, and 20% possessed the SPM gene. This study is one of the first to show what Pseudomonas aeruginosa is made of in the Diwaniyah Governorate. It also shows how resistant it is and what genes are responsible for making beta-lactamase. Conclusion: The results show how important it is to keep an eye on things all the time, follow strict infection control rules, and have antimicrobial stewardship programs in place to stop the spread of drug-resistant Pseudomonas aeruginosa in healthcare settings.