Currently, clinical laboratories lack an effective method to differentiate between classical Klebsiella pneumoniae (cKP) and hypervirulent Klebsiella pneumoniae (hvKP) strains, leading to delays in diagnosing and treating hvKP infections. Previous studies have identified peg-344, iroB, iucA, prmpA, prmpA2, and siderophores (SP) yields greater than 30 μg/ml as reliable markers for distinguishing hvKP from cKp strains. However, these diagnostic tests were conducted on a relatively small study population and lacked sufficient clinical data support. In this study, hvKP strains were identified by biomarker analysis and the Galleria mellonella model. Combined with in vitro and in vivo experiments, the reliability of clinical identification method of hvKP was verified, which provided an experimental basis for timely diagnosis of hvKP infection.According to the clinical data, a total of 108 strains of hvKP were preliminary screened. Among them, 94 strains were further identified using PCR analysis of biomarkers and quantitative determination of SP. The high virulence of hvKP was subsequently confirmed through infection experiments on Galleria mellonella. Additionally, susceptibility testing revealed the identification of 58 carbapenem-resistant hvKP (CR-hvKP) strains and 36 carbapenem-sensitive hvKP (CS-hvKP) strains. By comparing molecular diagnostic indexes, molecular characteristics such as high SP production of CR-hvKP were found.The combination of clinical data and molecular diagnostic index analysis effectively enables the identification of hvKP, particularly CR-hvKP. This study provides a scientific basis for accurate clinical identification and timely treatment of hvKP.© 2023. The Author(s).