Fatty acid (FA) analysis is a tool for dietary investigation that complements traditional stomach content analyses. Controlled feeding experiments were used to determine the extent to which the FA composition of diet is reflected in the liver and muscle tissue of the Port Jackson shark Heterodontus portusjacksoni. Over 10 wk, two groups of sharks were fed prawns or squid, which have distinct FA profiles. The percentage of total FA was significantly different for shark liver and muscle tissue when comparing controls with prawn- and squid-fed sharks. Compared with experimentally fed sharks, control shark muscle and liver had higher levels of 18:1n-9 and 20:2n-9. When comparing prawn- and squid-fed sharks, only liver tissue showed a significant difference in FA profiles. The livers of prawn-fed sharks were comparatively higher in 18:1n-7, 22:5n-3, 20:0, and 18:1n-9, while the squid-fed sharks had higher levels of 16:0 and 22:6n-3. These FAs in shark liver tissue were all reflective of higher amounts in their respective dietary items, demonstrating the conservative transfer of FA from diet to liver tissue. This study shows that liver and muscle FA profiles can be used as indicators of dietary change through the comparison of controls and fed sharks. The timescale of this study may not have been sufficient for capturing the integration of FA into muscle tissue because only liver FA profiles were useful to distinguish between sharks fed different diets. These findings have important implications for sampling design where FA profiles are used to infer dietary preferences.