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Next-generation sequencing is a well-established technique for sequencing of DNA and has recently gained attention in many fields of medicine. Our aim was to evaluate the accuracy of next-generation sequencing in identifying the causative organism(s) in patients with periprosthetic joint infection.In this prospective study, samples were collected from 65 revision arthroplasties (39 knees and 26 hips) and 17 primary arthroplasties (9 hips and 8 knees). Synovial fluid, deep tissue, and swabs were obtained at the time of the surgical procedure and were shipped to the laboratory for next-generation sequencing. Deep-tissue specimens were also sent to the institutional laboratory for culture. Sensitivity and specificity were calculated for next-generation sequencing, using the Musculoskeletal Infection Society (MSIS) definition of periprosthetic joint infection as the standard.In 28 revisions, the cases were considered to be infected; cultures were positive in 17 cases (60.7% [95% confidence interval (CI), 40.6% to 78.5%]), and next-generation sequencing was positive in 25 cases (89.3% [95% CI, 71.8% to 97.7%]), with concordance between next-generation sequencing and culture in 15 cases. Among the 11 cases of culture-negative periprosthetic joint infection, next-generation sequencing was able to identify an organism in 9 cases (81.8% [95% CI, 48.2% to 97.7%]). Next-generation sequencing identified microbes in 9 (25.0% [95% CI, 12.1% to 42.2%]) of 36 aseptic revisions with negative cultures and in 6 (35.3% [95% CI, 14.2% to 61.7%]) of 17 primary total joint arthroplasties. Next-generation sequencing detected several organisms in most positive samples. However, in the majority of patients who were infected, 1 or 2 organisms were dominant.Next-generation sequencing may be a useful adjunct in identification of the causative organism(s) in culture-negative periprosthetic joint infection. Our findings suggest that some cases of monomicrobial periprosthetic joint infection may have additional organisms that escape detection when culture is used. Further study is required to determine the clinical implications of isolated organisms in samples from patients who are not thought to be infected.Diagnostic Level I. See Instructions for Authors for a complete description of levels of evidence.