Background Appropriate training of health professionals has been recommended to increase organ donation rates. Some studies have shown insufficient knowledge among medical students. This survey aims to describe their knowledge and attitude toward organ donation (OD). Method We designed, pre-tested and conducted an online survey of all undergraduate medical students from Montreal, Laval and Sherbrooke universities in 2016–2017. Multivariate linear regression identified factors associated with a better knowledge score. Results Twenty-two percent (750/3344) of students completed the survey. Ninety-one percent of students adequately knew that neurological death is irreversible; 76% acknowledged that someone could be neurologically deceased while his heart is still beating; 69% were not aware of circulatory determination of death. For only one knowledge item, senior students had a better answer than junior students. Total knowledge score was associated with exposure to OD during medical studies and comfort in answering patients’ questions about OD (p < 0,001). Regarding attitude, 96% of respondents wished to become organ donors after death and 92% supported OD training during their medical training. Conclusion Despite a favourable attitude, medical students have limited knowledge of OD. Findings suggest the need for a formal curriculum about OD, as students expressed.