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Abstracts Background The purpose of this study was to compare the perception of upper dental midline deviation on the attractiveness of a smile among raters from different ethnicities, professions, genders, and ages and measure to what extent the presence or absence of the associated smiling structures influence the raters' evaluations. Methods A male subject (26 years of age) with adequate smile characteristics was selected by 3 experienced orthodontists, and 561 raters from 2 different ethnic groups (281 Chinese raters and 280 Black raters) rated the subject's smile after the subject's upper dental midline was digitally altered from 0 to 5 mm using a 5-point Likert scale on 12 smile photographs divided into two groups: group 1, in the presence of smile related structures, two-thirds of the nose, lips, and chin (NLC), and group 2, in the absence of smile related structures, the lips only (L). Results There were statistically significant differences (p < 0.05) between the two ethnicities, in 2 mm and 4 mm in-group NLC and 5 mm in-group L, as well as the raters' profession to each midline shift of both groups (NLC) and (L) for both ethnicities except for 0 mm. Regarding the role of associated smile structures, the smile photos were observed in the presence of smile-associated structures, and in its absence (NLC × L), statistically significant differences (p < 0.05) were found when the deviation was 5 mm among the Chinese raters; in 1 mm, and 4 mm among the Black raters. Among different genders, statistical differences were only reported (p < 0.05) for Chinese raters for 5 mm in NLC, while statistical differences were observed for 2 mm and 3 mm in NLC for Black raters. For age categories, differences were observed (p < 0.05) for 4 mm, 5 mm in NLC and 4 mm, and 5 mm in L for Chinese raters, while 5 mm in NLC and 1 mm in L for Black raters. Conclusion Perception of the upper dental midline deviations was influenced by the factors of ethnicity, profession, presence or absence of smile-associated structures, as well as the gender and age of the raters.