Zinc is essential for the functioning of many enzymes and plant processes and the malting process. Arbuscular mycorrhizal fungi (AMF) can improve zinc (Zn) uptake in the important cereal crop barley (Hordeum vulgare) on Zn-deficient soils. Here we investigated the impacts of Zn fertilisation and AMF on the yield and grain quality of malting barley cultivars. Five barley genotypes were inoculated or not with the AMF Rhizophagus irregularis, and grown in pots either fertilised with Zn or not. Measurements of Zn nutrition and yield were made for all cultivars. Further analyses of grain biochemical composition, including starch, β-glucan and arabinoxylan contents, and analysis of ATR-MIR spectra were made in two contrasting cultivars. Mycorrhizal colonisation generally resulted in decreased biomass, but increased grain dimensions and mean grain weight. Barley grain yield and biochemical qualities were highly variable between cultivars, and the ATR-MIR spectra revealed grain compositional differences between cultivars and AMF treatments. Mycorrhizal fungi can affect barley grain Zn concentration and starch content, but grain biochemical traits including β-glucan and arabinoxylan contents were more conserved by the cultivar, and unaffected by AMF inoculation. The ATR-MIR spectra revealed that there are other grain characteristics affected by AMF that remain to be elucidated.