Abstract Background Metagenomic next-generation sequencing (mNGS) has been reported to improve pathogen identification in infectious diseases. This prospective cohort study aimed to explore the etiological diagnostic value of mNGS in peritoneal dialysis (PD)-related peritonitis. Methods Patients with PD-related peritonitis were consecutively recruited at the Nephrology Department of Nanjing Drum Tower Hospital. PD effluent samples for mNGS and culture were collected simultaneously. The positive rate, detection time, and consistency of mNGS and culture were compared. Results From August 1, 2021 to August 31, 2022, 38 patients with 41 episodes of PD-related peritonitis were enrolled. The positive rate of mNGS was higher than that of culture, although not statistically significant (92.7% vs 78.0%, P = 0.109). The average reporting time of mNGS was significantly shorter than that of culture (30.4 ± 10.5 vs 86.9 ± 22.2 h, P