Objective: To evaluate the penetration of root canal sealers into dentinal tubules when radiotherapy (RT) is applied to the head and neck region. Materials and methods: Ninety-six mandibular single-rooted premolars were randomly divided into two groups (n = 48). One group was not irradiated; the other was exposed to radiation 60 Gy (2 Gy/day, 5 days/week for 6 weeks). The root canals were prepared, and four subgroups were created based on the sealer used (n = 12): AH Plus (AHP), AH Plus Bio (AHPB), Bio-C Sealer (BC), and GuttaFlow Bioseal (GFB). One-millimeter-thick dentin slices were obtained from coronal, middle, and apical regions. Maximum penetration depth (MPD), penetration percentage (PP), and area (PA) values were evaluated using a confocal laser scanning microscope. Statistical analyses were performed using one-way ANOVA, Tukey HSD, and independent t-tests, with a significance level of α = 0.05. Results: Radiotherapy significantly increased the sealer PP in the middle-third of the AHP (p = 0.048) and AHPB (p = 0.022) groups, as well as in the apical-third (p = 0.028) of the GFB group. BC was not affected (p > 0.05). In all groups, penetration values increased from the apical toward the coronal-third. Among irradiated groups, the GFBRT showed higher PP (p = 0,037) in the apical and higher MPD (0,014) in the middle-third compared to BCRT Conclusion: Radiotherapy alters the dentinal penetration of sealers depending on material and root region. Clinical significance: The present findings contribute to the understanding of sealer penetration under simulated radiotherapy conditions and may serve as a basis for further clinical research.