The objective of this study was to determine if increased overjet (greater than 6 mm) influences the magnitude and reproducibility of natural smile and maximal smile in Caucasian adult females. Twenty adult females with an increased overjet (6–10 mm) and 20 control adult females (overjet 2–4 mm) with no history of orthodontic treatment volunteered to participate. The mean age in the control group was 30.1 ± 6.4 years and the mean age in the test group was 31.9 ± 10.8 years. Three-dimensional stereophotogrammetric images were captured of each subject for three expressions: at rest, natural smile, and maximal smile. The images were recorded twice on two separate occasions, 6 weeks apart. Images were landmarked and a partial ordinary Procrustes superimposition was used to adjust for the differences in head posture between the same expressions. The magnitude of movement relative to the rest position, averaged over all the landmarks, was calculated and compared between the groups using analysis of variance (linear mixed-effects model); the intra- and inter-session reproducibility of both expressions was assessed. There was greater mean movement, averaged over all the landmarks, in the control group than in the increased overjet group for both natural smile and maximal smile (P = 0.0068). For these expressions, there were no statistically significant differences in reproducibility within sessions (P = 0.5403) or between sessions (P = 0.3665). Increased overjet had a statistically significant effect on the magnitude of smiling but did not influence the reproducibility of natural or maximal smile relative to controls.