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Abstract Background There has been a lot of debate on genetically modified (GM) crops both globally and locally in Uganda. Whereas some of the debates have been informed by scientific research, many are not. The level of acceptance and attitudes of people towards GM crops is a function of their knowledge. However, there is a paucity of studies on the knowledge and attitudes of Ugandans on GM crops. This study aimed to assess the level of knowledge of GM crops in Uganda. Methods We carried out a mixed methods study in September 2021 in Bushenyi, Jinja and Wakiso districts. We conducted 18 focus group discussions (FGDs), 13 key informant interviews (KIIs) and 698 quantitative interviews. The quantitative interviews were conducted using structured questionnaires. The FGD and KI interviews were audio recorded and transcribed verbatim. The qualitative data were analyzed using framework analysis and the quantitative data were analyzed using modified Poisson regression to identify factors associated with the level of knowledge of GM crops in STATA 15. Results Out of the 698 respondents interviewed, only 273 (39.1%) had ever heard of GM crops. About 204 (74.7%) of the 273 respondents reported having a moderate–high understanding of GM crops and 62.3% (170) of the respondents further reported that GM crops are harmful to their health and environment, despite some of their intrinsic benefits, such as high productivity, improved income and resilience to pests and diseases. In addition, Out of the 698 respondents interviewed, only 37.7% were wary of the possibility of the emergence of super pests due to the development of resistance to some GM crops. Conclusions Most of the community members do not have adequate knowledge about GM crops, hence, there is need for sensitization and legislation on GM crops before their release to the public.