Purpose The purpose of this study was to determine the prevalence of C-shaped root canal system in mandibular first premolar in Chinese population and to analyze the anatomical characteristics by Cone-beam computed tomography (CBCT) and Micro–computed tomography (Micro-CT) together. Methods CBCT images of 760 patients (1520 mandibular first premolars) were evaluated for the prevalence of C-shaped root canal system and the correlation with gender, age and position. 66 mandibular first premolars with C-shaped root canal system were scanned by Micro-CT and analyzed for morphologic features of the root canal anatomy (radicular groove, C-shaped root canal categories, accessory and connecting canals, apical foramina and accessory foramina). Results C-shaped root canal system was found in 16.9% of mandibular first premolars. The minimum mesial wall thickness was mostly measured at the lingual site (69.7%). Among the cross-sectional C-shaped root canal system classifications, the highest prevalence was C2 type (41.5%). In the 3D C-shaped root canal system classification, the highest prevalence was S type (34.8%). Accessory canals were observed in 48.5% of the samples, mostly in the middle region. The presence of 1-3 variable connecting canals was observed in 42.4% of the samples. The number of apical foramina was predominantly 1 (40.9%). Conclusions The incidence of C-shaped root canal system in mandibular first premolars was 16.9% in the Chinese population. It mostly had anatomical features such as deep root surface grooves, with C2 type and S type being the most prevalent.