Because gluten‐free type brewing with unmalted sorghum does not provide adequate nitrogen for complete fermentation, wort supplementation with ammonia (as diammonium phosphate, DAP) or lysine on yeast performance was investigated. By Phenotype Microarray, under aerobic conditions, greater yeast growth was indicated with DAP than lysine both as a single source and combined with sorghum wort amino acids. With sorghum fermentation, both DAP and lysine improved maltose and maltotriose uptake. However, DAP supplementation also maintained yeast numbers (24.0–21.3 × 106 cells mL−1), whereas there was a decline with lysine supplementation. Lysine supplementation also resulted in adverse effects on yeast cell morphology. Neither DAP nor lysine supplementation resulted in evident genetic change to the yeast, but the change in substrate from barley malt wort to unmalted sorghum wort slightly altered the yeast genetically. Therefore, ammonia as DAP has potential as a nitrogen supplement for improving yeast fermentation performance in sorghum gluten‐free brewing.