Introduction: The aim of this study was to evaluate the prevalence and characteristics of C-shaped root canals in maxillary first (MFMs) and second (MSMs) molars in a Southwestern Chinese population using cone-beam computed tomographic (CBCT). Methods: CBCT images of MFMs (n = 1488) and MSMs (n = 1547) from 1508 subjects enrolled in Guiyang Hospital of Stomatology between July 2018 to February 2021 were evaluated for the incidence and types of C-shaped root canals. Differences by age, sex, left or right side, and bilateral symmetry were also evaluated. Results: C-shaped root canals were present in 2.93% MFMs and MSMs (n = 3035) in the Southwestern Chinese population. A significant higher incidence was observed in the MSMs (5.24%) than in the MFMs (0.54%). Increased incidences were noted in teeth with fused root. Two major types and 5 subtypes of C-shaped canal system of maxillary molars were defined, and the most common type of C-shaped canals is fusion of mesial-buccal (MB) and distal-buccal (DB) canals (Type I subtype C). No significant gender and age differences were noted in the prevalence of C-shaped root canals in this population, and no significant difference was seen in its incidence in the left or right MFMs and MSMs. The bilateral occurrence was observed in 1.27% of the patients. Conclusion: C-shaped root canals are more frequently present in MSMs than in MFMs in the Southwestern Chinese population, with Type I subtype C (MB-DB canal fusion) as the most common subtype and low incidence of bilateral symmetry.