16S rRNA gene sequence examination is an effective instrument for characterization of new pathogens in clinical specimens. Akey component of colonization, biofilm formation, and protection of the pragmatic human pathogen Pseudomonasaeruginosais the biosynthesis of the exopolysaccharide Psl.Extracellular polysaccharides,biofilm, are secreted by microorganisms into the neighboring environment and are significant for surface attachment and keeping structural safety within biofilms.Biofilm production is an important technique for the survival of P. aeruginosa,and its association with antimicrobial resistance represents a defy for patient therapeutics. The aim of the current research is to assess the antibiotic resistance manner and distribution of the pslA gene among biofilm producingP. aeruginosa isolates, which have beengained from some hospitals in Baghdad, Iraq. Twenty-five P. aeruginosa isolates were obtained fromDepartment of Biology, College of Science, University of Baghdad. TheP. aeruginosa isolates were recognized using standard bacteriological techniques. Drug susceptibility test was done by disk diffusion technique for all the isolates against five antimicrobial agents.DNA was extracted from twenty-fiveP. aeruginosa isolates, which were selected as being resistant to gentamicin using the polymerase chain reaction(PCR). A specific primer pair was used to amplify 16S rRNA by a conventional PCR technique. Biofilm development was measured by microtiter plate test. The results of 16S rRNA showed that all 25 selected isolates were resistant to gentamicin harbored this gene. Biofilm formation was observed in 24/25(96%) of the P. aeruginosa isolates. The possibility of biofilm formation was remarkablyrelatedtothe resistance to gentamicin. In addition, the pslA gene was existed in all biofilm and non-biofilm producing the selected isolates with a frequency of 100% (n = 25).16S rRNA sequencing can be used to identify genetically atypical P. aeruginosa isolates from different origins. Theresults of the currentresearch well clarified that the P. aeruginosa biofilm-forming isolates were more resistant to the tested antibiotics. What is more, because of wide spreading, it appears that the pslA gene is associated with biofilm formation.