Since soybean is widely cultivated around the world and has a high protein content, it is a great nutritional vehicle for increasing the dietary uptake of selenium (Se). Several studies have evaluated biofortification with Se through fertilizer application in several crops. However, it is not clear how each method and source affect the total Se content or Se species in soybean grains. This work aimed to assess the total Se content and Se speciation in Se-enriched soybean grains produced under different Se application methods in the field. The treatments consisted of Se application (soil or foliar), using organic or inorganic Se sources at 10 g ha−1 or 80 g ha−1, in two genotypes. The results showed that all treatments with inorganic Se (soil and foliar) increased the Se content in grains compared with the control. More than 80% of the total Se in grains was present as selenomethionine (SeMet), and the speciation was affected by the Se source and the method of application. The treatments using inorganic Se, applied via soil or foliar, produced the highest content of Se as SeMet in soybean grains. Finally, we propose that the preservation of the Se species in products derived from soybean grains be evaluated as the following step.

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