The purpose of this research was to develop a questionnaire to assess the multidimensional construct of teamwork in sport and to examine various aspects of validity related to that instrument. A preliminary questionnaire was first created, and feedback on this instrument was then obtained from a sample of team-sport athletes (n = 30) and experts in sport psychology (n = 8). A modified version of the questionnaire was then completed by 607 athletes from 48 teams, and 5 multilevel confirmatory factor analyses were conducted to examine the structural properties of data derived from this instrument. Evidence of adequate model-data fit along with measurement reliability was obtained for each of the 5 models. Taken together, the results from this research provide support for the content, substantive, and structural aspects of construct validity for data derived from the 66-item Multidimensional Assessment of Teamwork in Sport.