The purpose of this research was to explore the speciation and bioaccessibility of native soil-derived selenium (Se) versus Se applied via fertiliser in the edible portions of maize, groundnut and cowpea grown in Malawi. Fertiliser-derived Se, applied as isotopically labelled selenate, contributed 88–97% of the total Se in the edible portions. Both soil and fertiliser-derived Se were transformed into similar species, with more than 90% of the extracted Se in an organic form. The main form of fertiliser-derived Se in grain was selenomethionine with an abundance of 92.0 ± 7.6% in maize, 63.7 ± 6.2% in cowpea and 85.2 ± 1.9% in groundnut. In addition, cowpea contained 32.7 ± 6.2% of Se-methyl-selenocysteine. The mean bioaccessibility of fertiliser-derived Se was 73.9 ± 8.5% with no statistically-significant difference across all crops despite some variation in speciation. Understanding the contribution of fertiliser-derived Se to the formation of organic forms of Se in crops is crucial, given that organic Se species are more bioaccessible than inorganic forms.