SummaryThe purpose of this study was to analyze the sealing ability of different root canal filling materials over a 6‐week period using a glucose penetration model. Forty‐six recently extracted human premolars were used in this study. The root canals were enlarged to 40/0.06. Prepared canals were randomly assigned into four groups (n = 10) as follows: Group 1, Gutta‐Percha (GP)/AH Plus with cold lateral compaction; Group 2, GP/AH Plus with continuous wave compaction; Group 3, RealSeal SE obturation system; and Group 4, OrthoMTA. The remaining specimens were used as positive and negative controls, and all specimens underwent thermocycling (10,000; 5–55 °C). The sealing ability of all samples was evaluated at 24 h, 1, 2, 4, and 6 weeks using a quantitative glucose leakage model, and scanning electron microscope (SEM) images were taken. A mixed effect analysis using R statistical language was performed. Groups 1, 2, and 4 showed low leakage levels during experimental periods. Group 3 showed low leakage levels for the first 2 weeks; however, the leakage level was significantly increased after 2 weeks compared to negative control group (p < 0.05). In the SEM results, Group 3 showed imperfect dentin bonding, whereas Group 4 showed calcium silicate hydrate short tags, which are formed at the access of the dentin tubules. GP/AH Plus and OrthoMTA showed less microleakage than RealSeal SE obturation system when used as root canal filling materials. Traditional GP/AH Plus sealer and the newly developed OrthoMTA are more appropriate for ideal sealing of the root canals. SCANNING 37:237–245, 2015. © 2015 Wiley Periodicals, Inc.