Objective: To compare the root canal morphology in the left and right mandibular premolars on cone beam computed tomography (CBCT) images. Methods: In this analytical cross-sectional study, 457 CBCT images, including human left and right mandibular premolars were evaluated. CBCT scans were investigated in the three spatial planes and differences in the number of roots and canals were recorded. Data were analysed using chi-square test. The significance level was set at P 0.05. Results: From 914 mandibular first premolars, in the left side, 85.6% had one root and 14.4% had two roots; 69.6% had one canal and 30.4% had two canals. In the right side 87.3% had one root and 12.7% had two roots; 66.5% had one canal and 33.5% had two canals. There was no statistically significant difference in the number of roots (P = 0.440) and canals (P = 0.321) between left and right mandibular first premolars. From 914 mandibular second premolars, in the left side, 94.3% had one root and 5.7% had two roots; 77.9% had one canal and 22.1% had two canals. In the right side, 95.6% had one root and 4.4% had two roots; 83.4% had one canal and 16.6% had two canals. There was a significant difference in the number of canals between the left and right mandibular second premolars (P = 0.036). Conclusions: The root canal morphology in the mandibular left and right premolars was different, but there was a significant difference only in the number of canals in the mandibular second premolars. Keywords: cone-beam computed tomography, mandibular premolar, number of canals, number of roots, root canal morphology