Organ donation currently is an extremely important issue in public health. Proper information about the details of this topic is extremely important, but is not yet widespread among the public. This study was carried out with the aim to determine the level of knowledge about organ donation among medical students in Paraguay and associated socio-academic factors influencing their level of knowledge. This was an analytical cross-sectional study, based on a multicenter survey among university medical students. About 68.7% (235) of the respondents were preclinical students doing basic sciences. Two aspects were evaluated, the knowledge regarding the donation of the organs and the socio-academic factors, then both the aspects were evaluated through bivariate and multivariate analyses. There were 342 respondents with a median age of 22 years (interquartile range: 20-23 years) of which 263 (77%) were women. One hundred and eighty-eight (55%) reported not knowing the law that protects and regulates the activities of organ and tissue donation in Paraguay. In the multivariate analysis, the highest frequency of a good level of knowledge of organ donation occurred in those who were older [RPA: 1.07, 95% confidence interval (CI): 1.02-1.12, P = 0.007] and in two of the universities evaluated (both with values P <0.012). On the contrary, those who were preclinical students, in general, had a lower level of knowledge of organ donation (RPa: 0.61, 95% CI: 0.46-0.79; value P <0.001). Our findings denote relatively a poor knowledge of organ donation in some socio-academic subsets. Therefore, it is important to develop strategies to increase the knowledge about the subject, by creating opportunities by way of discussions and debates among the students at all academic levels and also by conducting academic conferences on the subject.