Abstract Stable isotope composition of organisms from different trophic groups collected from a semi‐isolated wetland pool in the Ross River estuary, northern Australia, was analysed to determine if there was a consistent relationship between δ 13 C, δ 15 N and trophic level that could be used to assign trophic positions. A strong linear negative relationship between δ 13 C and δ 15 N was detected for the three trophic levels considered (primary producers, primary consumers and secondary consumers). This relationship was consistent among trophic levels, differing only in height, that is, on δ 15 N values, which indicate trophic positions. A difference of 3.6–3.8‰ between trophic levels was present, suggesting a δ 15 N fractionation of approximately 3.7‰, a value slightly higher than the commonly assumed δ 15 N fractionation of approximately 3.4‰. The relationship between δ 13 C and δ 15 N was similar for invertebrate and fish primary consumers, indicating similar δ 15 N trophic fractionation for both groups, meaning trophic positions and trophic length could be reliably calculated based on either invertebrates or fish.