The housing of wild animals in managed care facilities requires attention to all aspects of husbandry. Diets of wild animals often differ in composition, consistency, and quantity when compared with those in managed care settings including zoos, rehabilitation facilities, and aquaria. It was hypothesized that dietary differences from wild versus managed care would be reflected in data of circulating fatty acids based on previous studies. The current study examined the effect of species and environment on fatty acid concentrations in two omnivorous species of chelonians: Eastern box turtles, Terrapene carolina carolina, and common snapping turtles, Chelydra serpentina, located in the wild and managed care. Whole blood was collected and placed on spot cards for analysis of 26 fatty acids in a total lipid fatty acid profile. The present research indicated that Eastern box turtles have significantly (P < 0.05) higher percentages of linoleic acid (18:2n6), eicosadienoic acid (20:2n6), and mead acid (20:3n9). Common snapping turtles have significantly (P < 0.05) higher percentages of myristic acid (14:0), dihomo-γ-linolenic acid (20:3n6), erucic acid (22:1n9), and n-6 docosapentaenoic acid (22:5n6). Environmental effects also were noted; wild turtles had higher percentages of α-linolenic acid (18:3n3), arachidic acid (20:0), eicosadienoic acid (20:2n6), and eicosatrienoic acid (20:3n3) (P < 0.05), whereas n-6 docosapentaenoic acid (22:5n6) was higher for the managed care group. Eicosadienoic acid (20:2n6), behenic acid; 22:0), adrenic acid (22:4n6), n-6 docosapentaenoic acid (22:5n6), and nervonic acid (24:1) were significantly different (P < 0.05) in species–environment interactions without any noted species or environment patterns. Fatty acids are useful for many important biological functions including proper immune system regulation, and therefore, the present research provides medically relevant data for reptile diagnostics. This research may help further improve diets of all chelonians kept in managed care, regardless of species.