The effects of different obturation techniques on calcium silicate-based sealer penetration in the presence of apically separated rotary files were evaluated. Forty-eight extracted mandibular incisors were used. ProTaper F2 rotary files were separated at the apical thirds. Samples were divided into four groups (n = 12) according to obturation technique used: (a) cold lateral compaction (CLC); (b) single cone; (c) bulk-fill (BF) without a core material; and (d) thermoplastic injection (TI). Specimens were sectioned horizontally at 1 and 3 mm from the apex and studied using a confocal scanning laser microscope. The maximum tubule penetration depth and percentage of penetration were measured. Data were statistically analyzed using parametric and nonparametric tests with a significance level of 5%. Regarding penetration depth, a significant difference was found at 1 mm (p < .05), while no significant difference was found at 3 mm (p > .05). At the 3 mm level, all of the obturation techniques showed similar penetration depths. Regarding penetration percentage, the values of the CLC and TI groups were statistically less when compared with the BF group at 1 and 3 mm levels, respectively (p < .05). Under the limitations of this in vitro study, results suggest that the obturation technique may present a significant effect on sealer penetration.