Objectives The separated root canal instruments may affect the quality of root canal filling, hence the success of endodontic treatment. The aim of this study was to evaluate the effects of separated file fragments of nickel-titanium rotary systems with different cross-section, taper and motion characteristics on the apical sealer penetration in oval-shaped root canals via confocal laser scanning microscope (CLSM). Materials and methods Distal roots of 60 mandibular molars with oval-shaped root canals were randomly divided into 4 groups as follows: group 1, FlexMaster Nickel Titanium Rotary File System (NTRFS) (separated instrument: 30/.06); group 2, ProTaper Next NTRFS (X3); group 3, ProTaper Universal NTRFS (F3); group 4, Revo-S NTRFS (AS30/.06). Root canals were filled with gutta-percha and AH plus labelled with 0.1% rhodamine B using a warm vertical compaction technique. Each specimen was horizontally sectioned at 1st, 3rd and 5th mm from apical foramen. Amount of maximum and average penetration depths, penetration percentage and sealer penetrated area were measured and analysed with one-way repeated measures of ANOVA and the Bonferroni post hoc tests.p < 0.05 was considered significant. Results The penetration depth, percentage and penetrated area of the sealer increased from apical to coronal in all systems. The maximum and average penetration depths and penetration areas were higher in FlexMaster and Revo-S groups at the 3rd mm (p < 0.05). At the 5th mm, the Revo-S group had a higher penetration percentage, when compared with ProTaper Next and ProTaper Universal groups (p < 0.05). Conclusions In the 1st mm, separated fragments of any system did not allow the penetration of the sealer, while it was observed that the files with constant taper showed more positive results in terms of sealer penetration at apical 3rd and 5th mm.