We investigated the trophic ecology of whale sharks using carbon and nitrogen-stable isotope analysis from both whale shark sub-dermal tissue and an extensive set of potential prey including zooplankton and other pelagic organisms from Ningaloo Reef, Western Australia, in two consecutive years (2013 and 2014). Results showed that the composition of stable isotopes of whale shark sub-dermal tissue was not significantly different between years or sex. A positive relationship was however found between δ13C and δ15N and shark total length suggesting ontogenetic shift with size. When comparing whale shark sub-dermal tissue with potential prey isotopic signatures, δ15N values situated whale sharks as a secondary consumer similar to other zooplanktivores organisms. An enrichment in δ13C values in whale shark sub-dermal tissue resulted in no match in isotopic signatures between predator and potential prey. These results indicated that whale sharks might obtain part of their diet from other sources than merely pelagic. Overall, stable isotope analysis combined with non-lethal sampling techniques have demonstrated to be a good tool to examine trophic intravariability in large predators as well as to infer food web relationships.