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Public knowledge of medical genetics is essential for better establishment of its services but has been rarely evaluated based on distinguished types of knowledge. We designed and validated a new self-administered questionnaire in Farsi (Persian language) to assess public knowledge of medical genetics based on Rogers' framework. This framework divides knowledge into three types of awareness, how-to (practical) and principles knowledge which refer to knowing the existence, proper use, and theoretical principles of an innovation, respectively. We asked consecutive individuals (n = 306, age ≥ 20 years) visiting health centers in different regions of Yazd, a city in central Iran, to fill out the questionnaire. After validation, we analyzed 280 of the questionnaires which revealed a high degree of internal consistency (Cronbach's alpha 0.90) and a positive linear relationship among the scores of different knowledge. Our respondents had relatively fair awareness and how-to, but generally poor principles knowledge with statistically significantly better scores in females and those with higher education. We observed tangible strengths in topics such as consanguineous marriage, thalassemia, and hereditary predisposition to diabetes and cardiovascular disorders, and weaknesses in areas such as genetic testing and genetics of cancer. Notably, experience of premarital genetic counseling did not show any significant effect, but having a relative with a genetic disorder was significantly linked to better awareness scores. Our study provides a reliable and self-administered questionnaire for the assessment of public knowledge of medical genetics. Despite revealing important strengths and weaknesses in our population sample, larger scale evaluations in Iran and other developing countries are needed for better understanding of the public knowledge as the prerequisite for designing appropriate educational programs.