In this article, we present a useful method to identify malting barley varieties by genome analysis. To identify primer sets that permit the detection of polymorphism, various regions of genes of enzymes or proteins relevant to brewing were amplified by the polymerase chain reaction (PCR). The PCR-amplified fragments were analyzed by temperature gradient gel electrophoresis, and then sequenced. We developed the useful primer sets from the sequences of alpha-amylase, beta-amylase, beta-glucanase, and B1-hordein. The 10 typical barley varieties tested in this study were successfully distinguished by the restriction enzyme digest patterns of products amplified using these primer sets.