Abstract: A total of 1365 neonates with sepsis get admitted in the Chattagram Maa-O-Shishu Medical College Hospital during the surveillance period. Among them 8.35% (114) found bacteria borne neonatal sepsis with 98.25% Gram negative and 1.75% Gram positive bacteria. Klebsiella pneumoniae accounted for 69.30% (79), Serratia marcescens 16.67% (19), Acinetobacter 7.02% (8), Pseudomonas aeruginosa 3.51% (4), Proteus mirabilis 1.75% (2) and Staphylococcus aureus accounted for 1.75% (2). Most of the Gram negative bacteria showing resistance to commonly used antibiotics (Ampicillin, Ceftriaxone and Gentamycin) but sensitivity to the Imipenem. Klebsiella pneumoniae is the predominant pathogen in this study, resulting in further study like ESBL and molecular pattern of resistance of the isolated Klebsiella pneumoniae. Present Study found that as a whole 85% (67) of Klebsiella pneumoniae were multi-drug resistant out of 79 strains. The 86% (68) Klebsiella pneumoniae detected as ESBL producer, whereas, 14% (11) revealed ESBL non producer out of 79 isolates. This study only detected the occurrence of plasmid in Klebsiella pneumonia. The bands were failed to separate intensely on the gel due to very similar size of plasmids between them. Pulsed Field Gel Electrophoresis (PFGE) was done to determine the relationship among the different strains of Klebsiella pneumoniae. Three isolated strains were analyzed to perceive the clonal relation between the strains. All the three strains gave the same patterns of band on PFGE. That means all the three strains have similar type of DNA sequence. It indicates that, these three strains causing neonatal sepsis infection may be originated from the same clone.