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Aim: To determine the microorganism in root canal systems of root filled teeth with periapical disease and their relationship with clinical symptoms using nextgeneration sequencing. Methodology:The roots of 10 extracted teeth were collected from 10 patients who presented with post-treatment apical periodontitis (PTAP; six with symptoms and four without symptoms). Each root was divided horizontally into two parts (apical and coronal segments) and cryo-pulverized. Microbial communities were detected using 16S rDNA hypervariable V3-V4 region. The diversity, principal coordinate analysis and linear discriminant analysis effect size were performed in the symptomatic and asymptomatic groups (apical and coronal parts respectively). A Mann-Whitney test and an analysis of similarities were applied for intergroup analysis, at a significance level of 5%.Results: A total of 23 phyla, 257 genera and 425 species were detected. Firmicutes was the most abundant phylum in all samples. Three phyla (Fusobacteria, Synergistetes and unidentified_Bacteria) and seven genera (Fusobacterium, Porphyromonas, Phocaeicola, Olsenella, Campylobacter, Tannerella and Fretibacterium) were significantly more abundant in the symptomatic patients (p < .05), whereas asymptomatic patients had more Sphingomonas. The species more significantly abundant in the symptomatic samples were Porphyromonas gingivalis, Phocaeicola abscessus, Campylobacter showae, Tannerella forsythia and Olsenella uli (p < .05).Conclusions: A greater microbial diversity was observed in root filled teeth with PTAP compared to earlier reports. Several genera and species in root canal systems might be associated with clinical symptoms of PTAP.