The aim of this study was to evaluate root development in autotransplanted teeth using cone-beam computed tomography (CBCT) images. Twelve premolars with incomplete root formation, which were selected to replace prematurely lost upper central incisors, were analyzed by CBCT on two different occasions. The first CBCT examination (T1) was conducted before tooth autotransplantation. The second CBCT examination (T2) was performed over a follow-up period of at least 12 months and < 5 years. Three previously calibrated evaluators assessed root development. The positions of the tomographic planes were standardized. The mean root length in sagittal and coronal tomographic sections was used to validate the root length at T1 and T2. Longitudinal root development of the transplanted tooth was determined by calculating the difference in root length between T2 and T1. The intraclass correlation coefficient (ICC), paired t-test, and Pearson test were applied, with significance set at 5%. The mean time elapsed between T1 and T2 was 962 days/2.6 years. The ICC was > 0.75. The measurements obtained at T2 were significantly greater than at T1 (p = 0.001). The mean increase in root length was 2.83 mm. There was no significant correlation (p = 0.413; r = 0.261) between root length increase and the time elapsed between T1 and T2. Premolar teeth with incompletely formed roots transplanted to the upper central incisor region showed continued root development during postoperative follow-up.