Objective To characterize the molecular profiles of Klebsiella pneumoniae isolates causing neonatal sepsis and assess their clinical implications. Methods Clinical data and bloodstream isolates from neonates with Klebsiella pneumoniae sepsis admitted to four hospitals in Hebei Province from 2020 to 2024 were collected. Whole-genome sequencing was performed to analyze molecular characteristics of the pathogenic strains. Clinical features were compared between virulence gene-carrying and non-carrying groups. A phylogenetic tree based on sequencing data was constructed to explore potential transmission routes. Results A total of 37 infants and their clinical bloodstream isolates were included. Thirty-three (89%) had late-onset infection, and 32 (86%) were hospital-acquired. Preterm infants accounted for 95%. Twenty-eight (76%) underwent invasive procedures, and 13 (35%) had prior exposure to broad-spectrum antimicrobials. All infants were cured after treatment. Resistance rates to third-generation cephalosporins and carbapenems were 89% and 27%, respectively. ST45 was the most common sequence type. The carriage rates of extended-spectrum β-lactamase and carbapenemase genes were 84% and 24%, respectively. Concordance between resistance genes and phenotypic resistance was 86%. Clinical antimicrobial use was not fully consistent with resistance phenotypes or gene carriage. Among virulence genes, ybt had the highest carriage rate (54%). The proportion of concurrent infections at other sites did not differ significantly between the virulence gene-carrying and non-carrying groups (50% vs 29%, P=0.315). Phylogenetic analysis revealed intra-hospital and inter-hospital transmission of Klebsiella pneumoniae strains causing neonatal sepsis. Conclusions Detection of antimicrobial resistance and virulence genes facilitates deeper understanding of bacterial pathogenesis and provides a basis for optimizing regional infection control and antimicrobial treatment strategies.