Northern tuatara Sphenodon punctatus punctatus survive on about 26 islands off the north-east coast of the North Island, New Zealand. These rare, lizard-like reptiles include seabirds in their diet. The presence of the Pacific rat Rattus exulans on some islands has been implicated in the decline of tuatara populations through predation and competition for food. Pacific rats and tuatara eat similar dietary items, including seabirds. Seabirds have high levels of the long chain n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The potential contribution of seabirds to the diet of tuatara was investigated by measuring the composition of plasma fatty acids and the concentrations of triacylglycerol and cholesterol of adult tuatara from Green Island (rat-free, numerous seabirds) and Coppermine Island (rat-inhabited, relatively few seabirds). The composition of plasma fatty acids differed between islands and sexes. Specifically, tuatara from rat-free Green Island had higher levels of the long chain PUFAs arachidonic acid, EPA and DHA compared to tuatara on rat-inhabited Coppermine Island, and males had significantly higher levels of DHA than females on both islands. Concentrations of plasma triacylglycerol and cholesterol were higher in tuatara from Green Island than Coppermine Island. Males had higher cholesterol but lower triacylglycerol concentrations than females. Differences in fatty acid composition and lipid concentrations between groups of tuatara imply differences in diet. Tuatara on the rat-free island evidently had a higher intake of n-3 PUFAs compared with tuatara on the rat-inhabited island. This is probably due to higher seabird consumption, or perhaps to the presence of other dietary items containing EPA and DHA. High concentrations of plasma triacylglycerol and cholesterol may be the result of a high intake of insect larvae. We recommend the direct investigation of the diets of northern tuatara and the consequences of differences in the composition of plasma fatty acids for reproduction.