Although fruit canopy position is known to affect phytochemical composition in a number of commercial crops, there is limited information on its impact on the nutritional quality of native Australian fruit. This study is the first to quantify ascorbic acid in tuckeroo (Cupaniopsis anacardioides) fruit and investigate the impact of canopy position in this species. High levels of ascorbic acid were found in the skin (mean of 423 ± 61 mg/100 g on a dry weight basis) and arils (60.0 ± 18.8 mg/100g), but not in the seeds (mean of 15.6 ± 4.3 mg/100g). The tree, side and height all significantly affected fruit mass, with larger fruit located on the northern bottom side of the canopy. Skin ascorbic acid content also varied significantly with the tree (responsible for 50.8% of the total variance observed in vitamin C content), canopy height (accounting for 0.9% of the total variance), with a marginal impact of the side (compass direction). Fruit from the top of the southern side of the tree typically had the highest ascorbic acid content. This inter-tree and within-canopy variation in the nutritional content of C. anacardioides fruit may have implications for sampling protocols and potential harvesters of this fruit.