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Objectives: Identification of the underlying pathogens of infective endocarditis (IE) is critical for precision therapy. Methods: We evaluated a metagenomic method with next-generation sequencing (NGS) for the direct detection of pathogens from the resected valves of 44 IE patients and seven rejected IE patients according to the modified Duke criteria. Results: NGS displayed sensitivity, specificity, positive predictive values and negative predictive values of 97.6%, 85.7%, 97.6%, and 85.7% compared with 46.2%, 100%, 100%, and 12.5% for blood culture and 17.1%, 100%, 100%, and 17.1% for valve culture and 51.4%, 100%, 100%, and 26.1% for valve Gram staining, respectively. Conclusions: NGS technique had superior sensitivity and shorter turnaround time compared with culture-based methods for identifying causative pathogens of IE. The NGS technology should be considered an essential supplement to culture-based methods, particularly for unculturable or difficult-to-culture microorganisms. Keywords: Metagenomics shotgun, Next-generation sequencing, Infective endocarditis, Diagnosis