Estuaries provide critical habitat for a vast array of fish and wildlife but are also a nexus for core economic activities that mobilize and concentrate contaminants that can threaten aquatic species. Selenium (Se), an essential element and potent reproductive toxin, is enriched in parts of the San Francisco Estuary (SFE) to levels known to cause toxicity, yet the risk of Se to species that inhabit the SFE is not well understood. We quantified Se concentrations in muscle, liver and ovary of the demersal cyprinid Sacramento Splittail from six regions in the SFE at three time points to evaluate Se exposure risk. Selenium levels exceeded proposed EPA criteria in ovary and thresholds of concern for liver in 15% and 20%, respectively, of fish collected in the fall of 2010, preceding the discovery of juvenile Splittail displaying a high incidence (>40%) of spinal deformities characteristic of Se toxicity, and again in 2011. No exceedances were detected in muscle tissue. Selenium concentrations varied significantly among regions for muscle (F_5,113 = 20.49, p < 0.0001), liver (F_5,113 = 28.4, p < 0.0001) and ovary (F_5,112 = 19.3, p < 0.0001) but did not vary between the wet and dry years, nor were they influenced by foraging trophic level or prey selection. Foraging location along the salinity gradient, defined by δ³⁴S values, explained regional Se exposures in Splittail. Relationships between tissues varied among regions for muscle and liver and muscle and ovary, but a single global relationship could be defined for ovary and liver Se concentrations. Our results suggest that the proposed EPA Se criteria for muscle tissue in Splittail may be under-protective as it would not have predicted exceedances in liver or ovary tissue and that the relationship between muscle tissue and ovary and liver may be Se concentration and seasonal dependent.