OBJECTIVES: The objective of this study was to evaluate the effectiveness of clinically applicable diagnostic methods such as Direct Vision (DV), Dental Operating Microscope (DOM), and two-dimensional (2D) radiographs in detecting canal orifices and root canal configurations in mandibular first premolars with deep radicular grooves. The study also aimed to compare these methods with micro-computed tomography (micro-CT) to better understand their diagnostic capabilities and to assess the applicability of the Ahmed et al. (2017) classification system across different imaging modalities. METHODS: A total of 133 mandibular first premolars with deep radicular grooves were scanned using micro-CT. After scanning, two examiners, blinded to micro-CT results, accessed and examined the root canal orifices of the study samples under DV, and DOM. Further evaluation of the root canal configurations was undertaken using exploration and 2D radiographic images. A second round of relocation of canals was undertaken if the radiographic view showed the presence of a missed canal. The data were then compared with the original reconstructed micro-CT images. Descriptive statistical analysis, Kappa and chi-square tests were performed to compare between methods and examiners, and the p-value was set at 0.05. RESULTS: Except for three teeth, all samples exhibited multiple canals. Micro-CT data revealed a wide range of root canal configurations, with the most common being 1MFP1-2 (19.54%), followed by 1MFP1-2-3 (15.78%), 1MFP1-2-3-2 (5.26%), and both 1MFP1-2-4 and 1MFP1-2-1 configurations, each at 4.51%. For both examiners, using the DOM resulted in detection of more canal orifices compared to DV (p < 0.001). A number of root canal configuration types including 1MFP1-2 and 1MFP1-2-1 was identified using exploration and radiographic imaging. Both examiners identified additional canals in the relocation phase. A significant difference was found when the radiographic method is compared with micro-CT results (p < 0.001), in which the latter method showed more complex root canal configuration types. CONCLUSIONS: Mandibular first premolars with deep proximal grooves exhibit considerable variability and complexity in their root canal systems. The use of a dental operating microscope (DOM) significantly improves the detection of canal orifices compared to direct vision. The most frequently observed root canal configuration was 1MFP1-2. A high percentage of additional canals may be missed due to atypical locations of bifurcations along the root. While micro-CT imaging is not applicable in clinical settings, it remains a valuable reference tool in studies for enhancing our understanding of root canal morphology and guiding the development of clinically feasible diagnostic strategies.