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PURPOSE We establish an accurate and rapid diagnostic method for pathogen detection in infectious uveitis using nanopore metagenomic next-generation sequencing (NGS). METHODS In eight patients with suspected infectious uveitis, we prospectively compared the accuracy and time taken for pathogen identification between conventional diagnostic methods, such as cultures and PCR, and nanopore metagenomic NGS. RESULTS All causative pathogens were identified using nanopore sequencing, while only five of the eight patients were confirmed positive for a specific pathogen using conventional methods. The overall sample-to-answer turnaround time of nanopore sequencing was much shorter than that of conventional methods in the bacterial and fungal infection (mean, 17 h vs. 4d, respectively; P = .028). The pathogens could be detected even when the quantity or quality of DNA was not optimal. CONCLUSION Nanopore metagenomic NGS is a promising diagnostic tool that can rapidly and accurately identify the causative pathogen in infectious uveitis.