Limit dextrinase (LD) is a unique de-branching enzyme involved in starch mobilization of barley grains during malting, and closely related to malt quality. Genotypic variation of LD activity is controlled by genetic factors and also affected by environmental conditions. Correlation analysis between LD activity and four malt quality parameters showed that LD activity was positively correlated with diastatic power, Kolbach index and the quality of malt extract, while negatively correlated with viscosity. The structure-based association analysis demonstrated that HvLDI, a gene encoding limit dextrinase inhibitor, was a major determinant of LD activity and malt quality. The single nucleotide polymorphisms associated with LD activity could be used in early generation selection for barley breeding.