The conservation of imperiled species often depends upon conserving their prey. Graptemys flavimaculata (Yellow-blotched Sawback) is an imperiled turtle endemic to the Pascagoula River system of Mississippi, USA. We investigated diet variability of G. flavimaculata relative to site geography, sex, seasons, and size. We captured individuals from May to October in 2007 and 2008 at two distant sites. One site is an inland freshwater tributary (Leaf River, LR) and the other site is coastal and tidally influenced (Pascagoula River, PR). Feces from males (n = 68) and females (n = 74) were collected and analyzed. Using the Index of Relative Importance (IRI; 0–100 scale, 100 = only item present in all samples), we found that G. flavimaculata are primarily sponge specialists for both males (IRI_LR = 85, IRI_PR = 91) and females (IRI_LR = 84, IRI_PR = 65). For both sexes, between-site differences were more varied than within-site differences; for the latter, prey items were typically similar for males and females at a single site, but importance varied. Seasonal comparisons suggest that both LR and PR females shifted diets towards mollusks during the spring and fall, possibly associated with increasing energy and calcium for reproduction. Based on the frequency of wood fragments in feces, submerged deadwood appears important for prey species, and therefore, managers should conserve deadwood along riverbanks (i.e., mature riparian corridors). If channel maintenance is needed, deadwood should be moved toward banks rather than removed to conserve structure for prey species colonization.