Background: Digital twins have the potential to significantly improve the efficiency and sustainability of the agri-food supply chain by providing visibility, reducing bottlenecks, planning for contingencies, and improving existing processes and resources. Additionally, they can add value to businesses by lowering costs and boosting customer satisfaction. This study is aimed at responding to common scientific questions on the application of digital twins in the agri-food supply chain, focusing on the benefits, types, integration levels, key elements, implementation steps, and challenges. Methods: This article conducts a systematic literature review of recent works on agri-food supply chain digital twins, using a list of peer-reviewed studies to analyze concepts using precise and well-defined criteria. Thus, 50 papers were selected based on inclusion and exclusion criteria, and descriptive and content-wise analysis was conducted to answer the research questions. Conclusions: The implementation of digital twins has shown promising advancements in addressing global challenges in the agri-food supply chain. Despite encouraging signs of progress in the sector, the real-world application of this solution is still in its early stages. This article intends to provide firms, experts, and researchers with insights into future research directions, implications, and challenges on the topic.