Objective To evaluate the application value of a new biomarker PEG-344 in the identification of hypervirulent Klebsiella pneumoniae (KP) and classical KP. Methods KP isolated from blood specimens of patients with bloodstream infection in a hospital from 2020 to 2021 were collected, virulence test results of Galleria mellonella was as the gold standard for grouping, strains were divided into hypervirulent group and classical group; string test, wzi gene sequencing and virulence gene detection (_prmpA, _prmpA2, PEG-1631, PEG-589, PEG-344) were performed. Results A total of 36 KP strains were collected, according to Galleria mellonella virulence test results, KP strains were divided into hypervirulence group (n=16 strains) and classical group (n=20 strains); string test positive strains in hypervirulence group and classical group were 87.50% (14 strains) and 15.00% (3 strains) respectively; the sensitivity and specificity of detection of virulence gene PEG-344 were higher than those of classical independent biomarkers _prmpA and _prmpA2. Except one wxwh3 (wzi-2) strain in classical group was PEG-344 positive, results of other strains were completely consistent with the virulence grouping. Conclusion PEG-344 gene encoding metabolite transporter can more accurately identify hypervirulent strains, timely remind clinical rational drug use, and provide basis for clinicians to optimize diagnosis and treatment plan.