Background: Heart disease is the number one killer of both men and women in the United States, yet a comprehensive and evidence-based heart disease knowledge assessment is currently not available. Purpose: This paper describes the two-phase development of a novel heart disease knowledge questionnaire. Methods: After review and critique of the existing literature, a questionnaire addressing five central domains of heart disease knowledge was constructed. In Phase I, 606 undergraduates completed an 82-item questionnaire. In Phase II, 248 undergraduates completed a revised 74-item questionnaire. In both phases, item clarity and difficulty were evaluated, along with the overall factor structure of the scale. Results: Exploratory and confirmatory factor analyses were used to reduce the scale to 30 items with ft statistics, CFI = 0.82, TLI = 0.88, and RMSEA = 0.03. Scores were correlated moderately positively with an existing scale and weakly positively with a measure of health literacy, thereby establishing both convergent and divergent validity. Discussion: The finalized 30-item questionnaire is a concise, yet discriminating instrument that reliably measures participants' heart disease knowledge levels. Translation to Health Education Practice: Health professionals can use this scale to assess their patients' heart disease knowledge so that they can create a tailored program to help their patients reduce their heart disease risk. (Contains 3 tables.)