<p>The aim is to compare periapical radiography and cone-beam computed tomography (CBCT) in assessing the number of root canals in mandibular incisors and in identifying untreated canals, using microcomputed tomography (microCT) as the reference standard. Images of forty-two mandibular incisors were obtained by microCT, periapical radiography, and CBCT. Five endodontist examiners independently analyzed the periapical and CBCT images to identify the presence of one or two canals. Subsequently, 10 teeth with two canals were selected and root canals obturated (5 teeth with only one canal obturated and 5 with both canals obturated). New periapical and CBCT images were acquired and the examiners identified any unfilled canals. Data analysis compared imaging methods with a significance level of 5%, using McNemar and chi-square tests. In pre-treatment assessment with periapical radiography, 40% of two-canal incisors were interpreted as having only one canal, showing a statistically significant difference (<i>p</i> = 0.002) compared to microCT images. CBCT correctly identified the number of canals in 85.5% and 85.9% of teeth with 1 and 2 canals, respectively, with no significant differences compared to microCT (<i>p</i> = 0.857). For post-treatment assessment, there were no statistically significant differences between periapical radiography and CBCT for the assessment of teeth with one (<i>p</i> = 0.145) or two (<i>p</i> = 0.099) filled canals. While 28% of the teeth with one filled canal was identified in CBCT, 48% of those were identified in periapical radiography. CBCT is superior to periapical radiography in detecting root canals in mandibular incisors before endodontic treatment. However, the diagnostic accuracy of periapical radiographs and CBCT images in post-treatment is impacted by the presence of obturation material.</p>