Purpose: Perception of maxillary midline diastema as an esthetic trait varies in relation to culture, age group, and racial background. Although midline diastema is accepted in Africa, there are few reports on interethnic perception of midline diastema among Nigerians. Methods: Participants aged 19 to 45 years were selected from six geographical zones of Nigeria; one state was randomly selected from each zone, and a local government area (LGA) selected from each state. A structured questionnaire including a set of color smile photographs with varying sizes of maxillary midline diastema (narrowest = 2 mm; widest = 6 mm) was administered. Information sought was age, gender, socioeconomic group, tribe, presence of a midline diastema, and grading of attractiveness of smile. This information was evaluated using a Likert scale. Results: A total of 402 subjects aged 19 to 45 years and a mean age of 36.3 years (± 0.714) took part in this study. The highest proportion of subjects with midline diastema was aged 21 to 30 years (33.9%), females (29.8%), from the Hausa ethnic group (35%), and from the class II socioeconomic group (31.9%). Among the ethnic groups, Hausa had the highest (65%) preference for midline diastema. Females (58.8%) and those in the class II socioeconomic group (68.1%) also had the highest preference for midline diastema. There was a strong relationship between incidence of and preference for midline diastema (P = 0.000). All ethnic groups rated maxillary midline diastema as attractive, especially when the width was within 2 to 3 mm (P = 0.02). Conclusion: All ethnic groups perceived maxillary midline diastema to be an esthetic attribute, provided the width was within 2 to 3 mm.