Little is known about the food safety knowledge of young adults. In addition, few knowledge questionnaires and no comprehensive, criterion-referenced measure that assesses the full range of food safety knowledge could be identified. Without appropriate, valid, and reliable measures and baseline data, it is difficult to develop and implement effective education efforts. Thus, the purpose of this study was to develop a comprehensive, valid, reliable food safety knowledge questionnaire. Questionnaire development followed this process: 1) use of published reports and input from experts in food safety and sanitation (n = 7) to identify key food safety concepts; 2) development of a question bank (n = 101) assessing knowledge of key concepts (i.e., cross contamination prevention, disinfection procedures; safe times/temperatures for cooking/storing foods; groups at greatest risk for food borne disease; foods that increase risk of food borne disease; and food borne disease pathogens); 3) refinement of initial questions by experts; 4) questionnaire pretest with young adults (n = 180) and refinement; 5) questionnaire pilot test (n = 126) and refinement; 6) final expert review and refinement; and 7) conversion into an on line survey. Young adults (n = 4343, mean age 19.9 [plus or minus] 1.SD years) from 21 universities and colleges across the country completed the questionnaire. Item analysis was used to determine the overall quality of the test and identify improvements needed. Livingston's coefficient of reliability for criterion-referenced tests was 0.92. The questionnaire met or exceeded generally recognized standards of reliability and validity. This questionnaire could be useful in baseline assessment of food safety knowledge and measurement of knowledge gained after an educational intervention in adults.