Background: The development of societies relies on the type and manner of activities performed at universities. Professors play a pivotal role in the development of universities and societies. Assessment of professors’ motivation and behavior and factors affecting them can improve the educational system and increase its performance. This study aimed to assess the validity and reliability of the Physician Teaching Motivation Questionnaire (PTMQ) from the viewpoint of clinical professors at Iran University of Medical Sciences (IUMS). Methods: This is an instrument psychometric study in which the questionnaire’s validity was determined through the face and content validity and its construct validity through exploratory and confirmatory factor analysis. Reliability was determined through the calculation of Cronbach’s alpha and intraclass correlation coefficient and consistency between agreers. The data were analyzed in SPSS 23 and LISREL 8.8. Results: The results showed that all 18 items had acceptable content validity (0.68-0.97). The Cronbach’s alpha and intraclass correlation coefficients were 0.81 and 0.86, respectively. The number of items remained unchanged at 18 according to exploratory factor analysis which classified them into six categories (intrinsic motivation, identified motivation, introjected motivation, career motivation, external motivation, and teaching assistant motivation) and explained 54% of the total variance of the mentioned variables. Also, the value of CFI was equal to 0.93 and the value of Cohen’s kappa was between 0.77 and 0.84. Conclusion: The Persian version of PTMQ is a valid and reliable instrument that can be used for research, educational, and practical purposes to evaluate the teaching motivation of clinical professors in other groups.