The stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹⁵N) are frequently used in food web studies as natural biological indicators for tracking ecological processes, interactions, and energy flows from the basal plant and detrital energy sources to the primary and secondary consumers. The spatial heterogeneity in the ecological, biogeochemical, hydrological, and geomorphological processes of aquatic ecosystems can influence the δ¹³C and δ¹⁵N isotopic baseline of food webs, which directly affect the isotopic composition of primary and secondary consumers. A better understanding of the significant drivers of δ¹³C and δ¹⁵N of aquatic organisms at the landscape scale would facilitate and improve the accuracy of the use of these isotopic ratios as natural tracers. The main objective of this study was to identify the leading mechanisms influencing the spatial variability of δ¹³C and δ¹⁵N across New Zealand aquatic ecosystems at the landscape scale. The aims of this study were; (1) to review and collate the findings from published stable isotopic studies that investigated the effect of environmental factors on δ¹³C and δ¹⁵N of aquatic organisms, (2) to identify the sources and scales of δ¹³C and δ¹⁵N variability for top-predatory fish across New Zealand lake environments and, (3) to identify the sources and scales of δ¹⁵N variability for top-predatory fish and invertebrates (predatory and non-predatory) across New Zealand stream and river environments. Lake and stream δ¹³C and δ¹⁵N data of aquatic organisms were collated from a range of published and unpublished scientific literature across New Zealand. Data from a total of 88 lakes sourced from 11 publications and 475 stream sites from 15 publications were assessed to investigate the direct and indirect effects of environmental factors on the spatial variability of stable isotopic ratios across lakes and streams. To do so, a combination of correlation analyses, principal component analysis (PCA), and piecewise structural equation modelling (pSEM) were carried out. The variability of top-predatory fish δ¹³C values across New Zealand lakes was best explained by the morphology of the lake. Fish sampled from lakes with large surface areas and volumes had higher δ¹³C signatures from lakes of smaller morphological size. I hypothesised that larger lakes allow for more influence of δ¹³C-enriched atmospheric CO₂ due to greater equilibration through gas exchange processes and turbulence regimes, contributing to the observed increase in enrichment of fish δ¹³C with lake surface area. The top-predatory fish δ¹⁵N most strongly reflected the watershed land use and showed no apparent relationship with lake size. The variability of non-predatory and predatory invertebrates, and top-predatory fish δ¹⁵N across New Zealand streams was best explained by the local land use in the upstream catchment and the nutrient concentrations, i.e., the total nitrogen and total phosphorus concentrations. Our findings suggest that δ¹³C and δ¹⁵N respond to different environmental drivers. Carbon isotopic ratios tend to reflect the natural ecosystem properties, whereas nitrogen isotopic ratios respond more readily to the watershed land use. The findings in this study were consistent with previous stable isotopic research conducted in North American and European aquatic ecosystems, which have examined the effects of environmental factors on C and N isotopic ratios among aquatic organisms. An enhanced understanding of the natural ecosystem processes and anthropogenic influences, contributing to the spatial distribution of δ¹³C and δ¹⁵N of aquatic organisms, can improve the study design and interpretation of stable isotopes as a tracer in food web and ecosystem ecology, by taking into account the natural and human-induced spatial and trophic subsidies.