Objective: To compare dental positional and gingival parameters of maxillary anterior teeth in unilateral cleft lip and palate (UCLP) after orthodontic treatment with canine substitution due to lateral incisor agenesis. Methods: This split-mouth study comprised 57 subjects with UCLP (31 male, 26 female) and agenesis of maxillary lateral incisor at the cleft side, from a single center. Canine substitution was completed after the secondary alveolar bone graft. Dental models were taken between 2 to 6 months after debonding (mean age: 20.4 years). The following variables were measured in the maxillary anterior teeth: crown height, width, proportion, and symmetry, as well as steps between incisal edges, gingival margins, tooth mesiodistal angulation and labiolingual inclination. Paired t-tests with Bonferroni post-hoc correction was used for comparisons between cleft and non-cleft sides (p<0.05). Results: At the cleft side, canines replacing missing lateral incisors had a higher crown height (0.77mm) and an increased width (0.67mm), and first premolars showed a shorter crown height (1.39mm). Asymmetries were observed in the gingival level of central and lateral incisors, with a greater clinical crown at the cleft side (0.61 and 0.81mm, respectively). Cleft side central incisors were more upright than their contralaterals (2.12º). Conclusions: Maxillary anterior teeth demonstrated positional, size and gingival height differences between cleft and non-cleft side after space closure of maxillary lateral incisor agenesis. Slight asymmetries in tooth position and gingival margin in the maxillary anterior teeth should be expected after orthodontic treatment in UCLP patients.