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Purpose: Peritonitis is the most important complication with high rate of morbidity and mortality in patients on continuous ambulatory peritoneal dialysis (CAPD) despite the success and advances. Rapid and accurate identification of pathogens causing peritonitis in a CAPD patient is essential for early targeted treatment. The aim of the study was to evaluate the role of 16S rRNA gene and ITS region PCR and sequencing in detecting bacterial and fungal pathogens from the dialysate of patients undergoing CAPD., Methods: Fifty eight peritoneal dialysate from suspected cases of peritonitis on CAPD were subjected to conventional culture as per the ISPD guidelines and automated culture system. A conventional PCR was performed to detect the 16S rRNA gene and ITS region. Sequencing and analysis were performed to identify the etiological agent from the remaining dialysate., Results: Among the 58 dialysate fluid, the etiological agents were identified in 8(14%) samples by conventional culture, 28(48%) by automated culture and 47(81%) by 16S rRNA sequencing and analysis. In 8 samples there was discordance in the results of the culture and 16S rRNA PCR. BLAST search of nine sequences obtained from 16S rRNA PCR revealed that these sequences matched best with uncultured bacterial clones. In eleven samples the sequence failed., Conclusion: The molecular tool 16S rRNA gene and ITS region PCR and sequencing cannot be used as a standalone test as it lacks sensitivity to identify some bacterial species due to high genetic similarity in some cases and inadequate database in GenBank. However, it could be used as a supplementary test to the culture method especially in the diagnosis of culture negative peritonitis., Competing Interests: Declaration of competing interest Nil., (Copyright © 2022 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)