We examined the trophodynamics of albacore tuna in the south-west Pacific Ocean using novel numerical and biochemical tracer approaches.   Specifically, General Additive Models (GAMs) were used as an exploratory and predictive tool to assess the influence of collection site and time, and individual length and age on bulk carbon and nitrogen isotopes and on fatty acid biomarkers.  Interpolated results were relayed on a landscape map of the south-west Pacific Ocean to show the spatial distribution of these biochemical tracers.  Clear spatial differences in δ¹⁵N and δ¹³C, and in fatty acid markers 20:5n3, 16:1n7 and 22:6n3 were detected and related to distinct biological (diatom vs dinoflagellates) and oceanographic (temperature and eddy) features off east Australia.  Stable isotopes with slower turnover rates, were shown to vary seasonally for carbon and annually for nitrogen, whereas fatty acids varied monthly and/or seasonally.  Ontogenetic patterns of biochemical parameters were also detected and related to migration and related feeding trends known to occur in the species. Detection of spatial and temporal trophic differences supports the use of stable isotopes and signature fatty acids as tools to detect within community changes over space and time.  Our findings also suggest that, these biochemical tracers can be used to monitor bottom and top order processes in respect to fisheries and climate change.