Aim The aim of the present study was to evaluate the in vitro comparative assessment of root canal centering ratio and canal transportation associated with ProTaper Universal (PTU) (Dentsply Maillefer, Ballaigues, Switzerland), ProTaper Next (PTN) (Dentsply Maillefer, Ballaigues, Switzerland), and ProTaper Gold (PTG) (Dentsply Tulsa Dental Specialties, Tulsa, OK, USA) rotary file systems, with or without glide-path preparation, using cone beam computed tomography (CBCT) analysis. Materials and methods A total of 120 mesial roots of extracted human mandibular first molar teeth were collected and randomly divided into three groups (n = 40) depending on the type of rotary file system used for mesiobuccal root canal instrumentation: Group 1: PTU, Group 2: PTN, and Group 3: PTG rotary file systems. Each group was further divided into two Sub-groups (a and b) with 20 specimens, depending on whether glide-path preparation was performed using the ProGlider (PG) file (Dentsply Maillefer, Ballaigues, Switzerland). Before the root canal instrumentation, mesiobuccal root canals of all specimens were first scanned using the NewTom Go CBCT machine (Cefla S.C., Imola, Italy), and all root canals were then instrumented according to their groups and sub-groups. All rotary files were used according to their manufacturer's guidelines. Post-instrumentation, CBCT images of all specimens were taken with the same exposure parameters as those used in pre-instrumentation CBCT imaging. The distance between the external root surface and the internal canal wall was measured in both bucco-lingual and mesio-distal planes at 3 mm, 5 mm, and 7 mm levels of the mesiobuccal root canal of all specimens, comparing the pre-instrumentation and post-instrumentation CBCT scans for the evaluation of canal centering ratio and canal transportation using NewTom NNT software (Cefla S.C., Imola, Italy). Data were analyzed using one-way analysis of variance (ANOVA), multiple pairwise Tukey post-hoc tests, and Student's t-tests; a p-value ≤ 0.05 was considered statistically significant. Results Canal centering ratio was significant bucco-lingually and mesio-distally at 3 mm and 5 mm levels between Groups 1a, 1b, and 3a, 3b (p < 0.05). However, at 7 mm bucco-lingually, a significant difference was observed between groups 3a and 3b, and mesio-distally between Groups 2a and 2b (p < 0.05). Canal transportation was significantly bucco-lingually at 3 mm, 5 mm, and 7 mm levels between Groups 3a and 3b (p < 0.05). However, mesio-distally, no statistically significant difference (p > 0.05) was seen between the groups at all three levels of the root canal. Conclusion The use of the PG/PTN rotary file system showed the maximum canal centering ratio at all three levels of the root canal compared to the PTU and PTG rotary file systems, whether used with or without a glide-path. The PG/PTN rotary file system showed the least canal transportation at the 3 mm level, while at the 5 mm and 7 mm levels, the PG/PTU rotary file system showed the least canal transportation.