Genomic DNA from six euplasmic wheat-barley addition lines and from the wheat and barley parents were analyzed for the presence of alpha-amylase gene sequences, using plasmid DNAs from two different alpha-amylase cDNA clones as hybridization probes. Barley-specific alpha-amylase gene sequences were detected only in addition lines 1 and 6 carrying barley chromosomes 1 and 6, respectively. One cDNA clone probe hybridized strongly to sequences on chromosome 6 but not to those on chromosome 1. The other cDNA clone probe preferentially hybridized to sequences on chromosome 1. It is concluded that structural genes for alpha-amylases are located on barley chromosomes 1 and 6. alpha-Amylase genes on the same chromosome appear to be closely related to one another but substantially different from those on the other chromosome.