Oncorhynchus nerka occur both as anadromous sockeye salmon that spend most of their life in the ocean, and as non-anadromous kokanee salmon that remain in fresh water their entire lives. We assessed whether stable isotopes of sulfur (δ34S) in otoliths could be used to distinguish sockeye salmon and kokanee ecotypes that are otherwise difficult to identify when they share a common freshwater rearing environment. We also investigated the chemical link between salmon and their diet by measuring δ34S in various fish tissues (eggs, muscle, scales) and zooplankton. δ34S (mean±SE) in sockeye salmon eggs (18.7 ± 0.4‰) and marine zooplankton (20.5 ± 0.1‰) were enriched by 10–14‰ compared with kokanee eggs and freshwater zooplankton. δ34S in the otolith cores of sockeye salmon (19.2 ± 0.7‰) and kokanee salmon (5.3 ± 1.1‰) were similar to δ34S in marine and freshwater zooplankton, respectively, indicating that the core is derived from maternal yolk tissue and reflects the maternal diet. δ34S in the freshwater growth zone of otoliths did not differ significantly between sockeye (5.9 ± 1.1‰) and kokanee salmon (4.4 ± 1.2‰), and was similar to freshwater zooplankton. The mean difference between δ34S in the otolith core and first year of growth was 13.3 ± 1.4‰ for sockeye and 0.65 ± 1.3‰ for kokanee salmon. A quadratic discriminant function developed from measurements of δ34S in otoliths of known maternal origin provided perfect classification rates in cross-validation tests. Thus, sulfur isotope ratios in otoliths are effective in discriminating between anadromous and non-anadromous ecotypes of O. nerka.