Aim To assess the impact of mechanical overinstrumentation of root canals in extracted teeth on the development of dentinal microcracks in the apical portion of the root by means of micro‐computed tomographic (micro‐CT) analysis. Root canal preparation short of the canal terminus and at the apical foramen (AF) were used as controls. Methodology Twenty 2‐rooted maxillary premolars with two canals were selected, scanned in a micro‐CT device and the root canals prepared sequentially using Reciproc M‐Wire R25 instruments to working lengths: 1 mm short of the AF (AF − 1 mm), at the AF (AF), and overinstrumentation (AF + 1 mm). A micro‐CT scan of each specimen was performed after each time point. After reconstruction and co‐registration procedures, the images were screened from the cementoenamel junction to the apex (n = 55 352) to identify the presence of dentinal microcracks in the apical third of the root. Results After the analyses of 55 352 slices, dentinal microcracks were visualized in 12.45% of the images (6892 slices), with 5.73% (3174 slices) in the cervical, 3.57% (1976 slices) in the middle and 3.15% (1740 slices) in the apical third. All dentinal microcracks observed at all time points (AF − 1 mm, AF and AF + 1 mm) were already present in the corresponding images before canal instrumentation. Therefore, no new microcracks were detected, regardless of the working length used for canal instrumentation. Conclusion Reciprocating root canal preparation either short, at or beyond the AF (overinstrumentation) did not create dentinal microcracks in the roots of extracted 2‐rooted maxillary premolars.