This study aimed to determine the incidence of dentinal microcracks after instrumenting the root canals of maxillary premolars using two novel rotary instrumentation systems. Micro-computed tomography (microCT) scans and images generated by sectioning and observation with a stereomicroscope were used to assess the specimens. Twenty-two freshly extracted maxillary premolars were collected and the specimens were divided into two groups of eleven. The tested radicular shaping systems were XP-Endo shaper and TRUShape (i.e., single file). The specimens were scanned with microCT pre- and post-instrumentation, and the newly formed microcracks were detected. The post-instrumentation scans were also compared with images obtained by sectioning method and stereomicroscope inspection, comparing the incidence of microcracks in either microCT scans or images. The results identified an overall incidence of 0.49% of newly formed microcracks, with no statistically significant differences (p = 0.689) between the shaping systems (0.11% for TRUShape and 0.87% for XP-Endo shaper). There were statistically significant differences (p < 0.001) between the microcracks incidence in microCT scans and the sectioning method (16.6% more for the latter). In conclusion, the results show that neither TRUShape nor XP-Endo shaper created dentinal microcracks during root canal instrumentation. The sectioning method with stereomicroscope evaluation overestimates the presence of microcracks with a statistically significant difference compared to microCT scans.