This study was to investigate root anatomy and root canal morphology of mandibular first premolars in a Chinese population. 178 human permanent mandibular first premolars extracted from a native Chinese population were collected, scanned using micro-computed tomography and reconstructed three-dimensionally. The number of roots and canals, canal configuration and radicular grooves were investigated. The root canal morphology was categorized according to Vertucci's criteria. The radicular grooves were scored according to the Arizona State University dental anthropology scoring system (ASUDAS), and the correlation between scores for radicular grooves and root canal morphology was analyzed. Almost all the samples were single-rooted (99.4%). 64.04% of teeth possessed type I canal systems, whilst 34.27% had two canals and 1.69% had three canals. According to ASUDAS, the scores of radicualr grooves were 56.74%, 16.85%, 12.36%, 10.11%, 3.37% and 0.56% respectively from grade 0 to grade 5. The roots with radicular grooves (grade 3 or 4) were defined as Tome's anomalous root and these roots have a high incidence of C-shape configurations (66.67%) and multiple-canal systems (100%). There is complicated variation of the root anatomy and canal morphology of mandibular first premolars in southwestern Chinese population, which needs special attention and careful assessment for endodontic treatment.