Limit dextrinase (LD) is a key enzyme in determining the malting quality. A survey of 60 barley varieties showed a wide range of variation for the enzyme activity and thermostability. Galleon showed low enzyme activity and high thermostability while Maud showed high activity and low thermostability. Alignment of the LD amino acid sequences of Galleon and Maud identified seven amino acid substitutions Lys/Arg-102, Thr/Ala-233, Ser/Gly-235, Gly/Ala-298, Cys/Arg-415, Ala/Ser-885 and Gly/Cys-888. Genetic diversity of LD was investigated using single strand conformation polymorphism based on the amino acid substitutions. Only limited genetic variation was detected in the current malting barley varieties, although wide variation was observed in the wider barley germplasm. The Thr/Ala-233 and Ala/Ser-885 substitutions were associated with enzyme thermostability ( < 0.0001), but no polymorphism was associated with the enzyme activity. This result was confirmed from further sequence analysis. The results will provide a tool for understanding and selection of high LD thermostability.