Abstract16S rRNA gene sequence analysis is a robust tool for Background: characterization of new pathogens in clinical specimens with suspected bacterial disease. The aim of this study was to characterize Pseudomonas isolated from clinical specimens by sequencing the 16S rRNA aeruginosa gene.Forty bacterial isolates were obtained from different clinical Methods: specimens (wound, urine and sputum) using enrichment selective media and biochemical tests to characterize and identify the bacteria as P. aeruginosa. DNA was extracted from using the Chelex method. A universal P. aeruginosa primer was used to amplify 16S rRNA genes by a conventional PCR technique. The amplified PCR products were sequenced, and the sequences were viewed by Finch TV program version 1.4.0. The identity and similarity of the nucleotide sequence of the isolated strains was detected by comparing them with published sequences using BLASTn. Phylogenetic trees were constructed using Phylogeny.fr software.Sequence analysis by BLASTn displayed high similarity and identity Results: with from China KX461910, Australia JN609194 and with other P. aeruginosa isolates from the GenBank database. P. aeruginosaOur observation of isolates from different origin sites, further Conclusions: show the utility of 16s rRNA PCR amplification. This reveals the high specify of the primers and accuracy of the PCR. Thus, 16S rRNA sequencing can be used to identify genetically atypical isolates from different origins. P. aeruginosa