Aim: The presence of a furcation groove on the palatal aspect of bifurcated maxillary first premolars (MFPs) leaves the dentine cementum wall (DCW) thickness critically thin in this region of the root. The purpose of this study was to evaluate the morphology of the furcation groove and the DCW thickness along the groove before and after root canal instrumentation, utilising cone beam computed tomography (CBCT). Materials and methods: Twenty-five human MFPs with bifurcated roots and furcation groove were divided into three groups according to one of three file systems: Hyflex CM, ProTaper Next (PTN) and Self-Adjusting File (SAF). Pre-instrumentation CBCT analysis was performed for the location, length, beginning and end of groove, and measurement of the minimum DCW thickness along the length of the groove. Post-instrumentation CBCT scans were analysed again for residual DCW thickness. The amount and percentage of cut dentine were calculated. Results: The groove began at a mean distance of 1.88 ± 0.13 mm above the bifurcation in 16 out of 25 samples, with a mean length of 5.61 ± 0.38 mm and a mean maximum depth of 0.7 ± 0.15 mm, located at a mean distance of 4.92 ± 0.15 mm above the apex. Preoperatively, the mean DCW thickness was 0.73 ± 0.07 mm. Post-instrumentation, statistically significant reductions in DCW thickness were observed with Hyflex 40/4% (P value = 0.0002), PTN X3 and X4 (P = 0.0002 and 0.0001) and SAF (P = 0.0002). Conclusions: Instrumentation of buccal canals of MFPs with furcation grooves should be performed using files with smaller tip sizes and minimum taper. Keywords: cone beam computed tomography, furcation groove, maxillary first premolar, residual dentine cementum wall thickness