Objective: To evaluate the effect of indirect anchorage with mini-screw assisted clear aligner on molar distalization. Methods: Twenty-six adult patients [12 males and 14 females, aged 19 to 31 years, mean age (26.4±4.2) years], who were treated in the Department of Orthodontics, the Affiliated Stomatological Hospital of Nanchang University between January 2018 and September 2021, were included. All the patients were treated with clear aligner and mini-screw indirect anchorage to distalize maxillary molars. In the stage of molar distalization, the mini-implants were implanted between the buccal roots of the maxillary first molar and the second premolar, and rigidly connected to the maxillary second premolar as anchorage reenforcement. Until the first molar had distally moved to the designated position, the mini-implants were transferred to the buccal area between the first and the second molars and similar device was connected to the first molar. Cone-beam CT (CBCT) images were taken before the treatment (T0), at the maxillary first molar distally moved to the designated position (T1), and at the anterior teeth alignment finished (T2). The CBCT data were imported into Mimics 20.0 software for three-dimensional reconstruction. After correcting the head position, each reference point was determined in the multi-plane reconstruction view. The bilateral porion, the right orbitale, the nasion, and the anterior nasal spine were selected as reference points to construct the three-dimensional space coordinate system. The positions of the first molar, the second premolar, and the incisor were analyzed three-dimensionally. The three-dimensional position changes of maxillary first molars and maxillary second premolars during the stages of T0-T1, T1-T2, T0-T2, and the mesiodistal and vertical position changes of maxillary central incisors were analyzed. The statistical analysis was carried out using a one-way repeated measures ANOVA. Results: There was a significant difference in the sagittal position changes of the root and cusp of the maxillary central incisors among three time points (F=24.84, P<0.001; F=27.66, P=0.001), but no difference was observed between T0 and T1 (P>0.05). There was a significant difference in lingual retraction of the maxillary central incisor root [(2.17±0.42) mm] and cusp [(1.81±0.28) mm] between T1 and T2 (P<0.05). A significant difference was noted in the sagittal position changes of the root and crown of the maxillary second premolars among three time points (F=17.16, P=0.001; F=57.99, P<0.001). However, no statistical difference was detected between T0 and T1 (P>0.05) and the difference in maxillary second premolar distalization [root (1.95±0.42) mm, cusp (2.53±0.33) mm] was observed between T1 and T2 (P<0.05). There was a statistically significant difference in the sagittal position changes of the root and the crown of the maxillary first molar among three time points (F=9.37, P=0.002; F=140.26, P<0.001). The difference in the maxillary first molar distalization [crown (3.51±0.30) mm, root (1.98±0.25) mm] between T0 and T1 was significant (P<0.05). However, no significant difference in the sagittal position of the maxillary first molars was observed between T1 and T2 (P>0.05). There was a statistically significant difference in the vertical position of the center point of the root in maxillary first molar among three time points (F=59.06, P<0.001), while (2.28±0.24) mm of intrusion between T0 and T1 was exhibited (P<0.05). However, no significant difference was found in the vertical position of the maxillary first molars between T1 and T2 (P>0.05). There was no significant difference in other measurements among three time points (P>0.05). Conclusions: The effect of indirect anchorage with mini-screw-assisted clear aligner on molar distalization was positive.