Background: Heart disease knowledge is an important concept for health education, yet there is lack of evidence on proper validated instruments used to measure levels of heart disease knowledge in the Malaysian context. Methods: A cross-sectional, survey design was conducted to examine the psychometric properties of the adapted English version of the Heart Disease Knowledge Questionnaire (HDKQ). Using proportionate cluster sampling, 788 undergraduate students at Universiti Sains Malaysia, Malaysia, were recruited and completed the HDKQ. Item analysis and confirmatory factor analysis (CFA) were used for the psychometric evaluation. Construct validity of the measurement model was included. Results: Most of the students were Malay (48%), female (71%), and from the field of science (51%). An acceptable range was obtained with respect to both the difficulty and discrimination indices in the item analysis results. The difficulty index ranged from 0.12.0.91 and a discrimination index of . 0.20 were reported for the final retained 23 items. The final CFA model showed an adequate fit to the data, yielding a 23-item, one-factor model [weighted least squares mean and variance adjusted scaled chi-square difference = 1.22, degrees of freedom = 2, P-value = 0.544, the root mean square error of approximation = 0.03 (90% confidence interval = 0.03, 0.04); close-fit P-value = > 0.950]. Conclusion: Adequate psychometric values were obtained for Malaysian undergraduate university students using the 23-item, one-factor model of the adapted HDKQ.