This study aimed to analyze the root anatomy and root canal system morphology of mandibular first premolars in a Chilean population. 186 teeth were scanned using micro-computed tomography and reconstructed three-dimensionally. The root canal system morphology was classified using both Vertucci’s and Ahmed’s criteria. The radicular grooves were categorized using the ASUDAS system, and the presence of Tomes’ anomalous root was associated with Ahmed’s score. A single root canal was identified in 65.05% of teeth, being configuration type I according to Vertucci’s criteria and code 1MP1 according to Ahmed’s criteria. Radicular grooves were observed in 39.25% of teeth. The ASUDAS scores for radicular grooves were 60.75%, 13.98%, 12.36%, 10.22%, 2.15%, and 0.54%, from grade 0 to grade 5, respectively. The presence of Tomes’ anomalous root was identified only in teeth with multiple root canals, and it was more frequently associated with code 1MP1–2 of Ahmed’s criteria. The root canal system morphology of mandibular first premolars showed a wide range of anatomical variations in the Chilean population. Teeth with multiple root canals had a higher incidence of radicular grooves, which were closely related to more complex internal anatomy. Only teeth with multiple root canals presented Tomes’ anomalous root.