Germplasms consisting of 64 wild barley accessions (Hordeum vulgare ssp. spontaneum) were examined for polymorphisms in α-amylase using both isoelectric focusing (IEF) and thermostability assays. Wide variation was found for the high pI α-amylase with 20 IEF band patterns identified. Enzyme activity and thermostability assays showed large differences among α-amylase isoenzymes. Two wild accessions Tel-Shoket CPI 77146-32 and Afiq CPI 77128-41 showed superior enzyme activity and thermostability compared with commercial varieties such as ‘Baudin’, ‘Flagship’ or ‘Navigator’. The functionality of the Tel-Shoket allele was validated in backcross lines with ‘Flagship’ as the recurrent parent. The Tel-Shoket allele at the amy1 locus increased α-amylase thermostability at 75°C by 8.4% and α-amylase activity in kilned malt by 18.7%. The introgression of the wild allele also led to significant improvements in fermentability, hot water extract and viscosity. Gene sequencing showed that there are three single nucleotide polymorphisms in the Tel-Shoket amy1 sequence, which can be used as diagnostic markers for the selection of this allele in breeding programmes.