Black-backed jackals are medium-sized canids with an omnivorous and opportunistic diet, allowing them to persist across different landscapes and land uses in southern Africa. Their diet is influenced by both top-down factors, like the presence of larger carnivores, and bottom-up factors, such as prey size, abundance, behaviour, and habitat. Therefore, varying land uses—such as livestock farming, game ranching, or nature Reserves can significantly impact jackal diet and behaviour due to variations in management strategies and species presence. We examined the dietary response of jackals to three land uses in a localized region of South Africa: a nature Reserve with larger carnivores, a game farm without larger carnivores, and livestock farms in the semi-arid Karoo, South Africa. Using DNA metabarcoding of jackal scats, we identified prey species across these landscapes. Results showed significant variation in diet, with the jackals in the nature Reserve displaying a broad diet, while livestock and game farms diet were dominated by specific prey species, such as sheep on livestock farms and greater kudu on the game farm. Rodents were a significant dietary component for black-backed jackals across all land uses, highlighting their importance as a resource. Similarly, large ungulates (greater than 100 kg) consistently comprised a stable part of the jackal diet across different land uses. Seasonal shifts indicated that rodents served as a crucial resource during the dry season, while medium-sized ungulates saw increased consumption during the wet season. Notably on the livestock farm, the consumption of steenbok peaked during the autumn and summer collection periods, even surpassing that of sheep. This study highlights the behavioural flexibility of jackals, illustrating their ability to adapt their diets based on prey availability, habitat conditions, and interactions with other predators. Key words: blacked-back jackal, flexible diet, different land uses, DNA metabarcoding