We investigated the spatial, seasonal, and within-plant variation in total fatty acids (TFA) and fatty acid (FA) composition in the brown seaweeds Dictyota bartayresii sampled from Nelly Bay, Orpheus Island, and Kissing Point (Northern Queensland, Australia) and Dictyopteris australis sampled from Nelly Bay. Both species were present year round, but with no consistent seasonal pattern in plant size, and had an annualized mean content of TFA of 5 % dry weight (dw) that is among the highest recorded for seaweeds. For Dictyota, larger plants had a higher content of TFA and a higher proportion of polyunsaturated omega-3 FA (PUFA(n-3)) than smaller plants, while for Dictyopteris, the TFA content and the proportion of PUFA(n-3) were unrelated to plant size. TFA in Dictyota varied considerably between locations (~40 %) (3.5–5.5 % dw) and to a lesser degree between seasons (~10 %) with a lower content of TFA and higher proportion of PUFA(n-3) in winter. Dictyopteris had an ~10 % higher content of TFA in winter but with no seasonal pattern in the composition of FA. There was also within-plant variation in FA, as TFA decreased from the tips toward the base in both species, and in Dictyopteris, the tips had a higher proportion of saturated FA (SFA) and PUFA(n-3) than the base. The presence of site- and species-specific variation in the content and composition of fatty acids reinforces the need for detailed analyses of the biochemical profile of seaweed biomass, including their extracts or purified compounds, if the focus is nutritional applications based on lipids.