© Inter-Research 2016.We examined the feeding ecology of whale sharks by analyzing the signature fatty acids of their sub-dermal tissue and those of an extensive set of potential prey collected at Ningaloo Reef, Western Australia, in 2013, 2014 and 2015. Sub-dermal tissue of whale sharks was low in lipid content (4.0 mg g-1 dry mass) and dominated by phospholipids (72% of total lipids), with a calculated energy density of 18.7 kJ g-1 dry mass. There was significant intraspecific variability in fatty acid profiles of whale sharks, with cluster analysis identifying 4 distinct groups in 2013 and 5 groups in 2014. As this variability was not related to sex or size-class, we suggest that it may be attributed to differences in the feeding habitats used by these groups of whale sharks. Variation in dietary patterns was also observed between years, likely due to changes in the primary and secondary producers. Examination of food web interactions showed that fatty acid profiles of whale sharks and their presumed prey were significantly different, suggesting that sharks fed over a wide range of habitats, including deep waters. Our findings show that signature fatty acids of sub-dermal tissue can be used to examine broad trophic pathways and to identify spatial and temporal changes in the diet of these large and wide-ranging animals.