A Cross-sectional study was designed that include 150 clinical specimens of urine, sputum, wound and otitis media obtained from patients with urinary tract infections respiratory infections, surgical wound and otitis media infection attended to age range (6-75 years), who visited Hilla teaching hospital and Marjan medical city. The period extended from August (2020) to November (2020). To confirm the isolates of Klebsiella pneumonia was used automated Vitek 2 system use GN-ID cards which contained 64 biochemical tests, the results demonstrated that all 67 isolates were confirmed with ID massage confidence level ranging excellent (probability percentage from 94 to 99.7. One sample was included, which showed about 337 bp amplicons length. Before sending these amplicons to sequencing, it was made sure that all the amplified amplicons had shown sharp, specific, and clean bands. The sequencing reactions had indicated the confirmed identity of the amplified products by performing NCBI blastn. Concerning the 337 bp PCR amplicons of the currently targeted entB sequences, the NCBI BLASTn engine showed a high sequence similarity between the sequenced sample and Klebsiella pneumoniae sequences. NCBI BLASTn engine indicated the presence of about 99% of homology with the expected target that covered a portion of the entB gene sequences. This genetic fragment is responsible for encoding on enterobactin biosynthesis bifunctional isochorismatase/aryl carrier protein EntB, with a total length of 283 amino acids. By comparing the observed DNA sequences of the currently investigated samples with the retrieved DNA sequences (GenBank acc. CP064352.1). After positioning the 337 bp amplicons’ sequences within the entB sequences, the details of these sequences were highlighted within the amplified sequences. The alignment results of the 337 bp samples revealed the detection of five nucleic acid variations compared with the corresponding K. pneumoniae referring sequences. These sequences were prepared by aligning our investigated samples with the most relative sequences deposited in the NCBI database (GenBank acc. CP064352.1).