A Chinese landrace of barley, Mokusekko 3, is completely resistant to all the present strains of Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV), and has at least two resistance genes, rym1 and rym5. However, rym1 has not been used for BaYMV-resistance breeding of barley in Japan so far. In our previous study, rym1 was completely resistant to BaYMV-I, -II, BaMMV-Ka1 and -Na1, and was acceptably resistant to BaYMV-III. Complete resistance of Mokusekko 3 was estimated to be due to the interaction between rym1 and rym5. To estimate pyramiding effects of these genes using DNA markers, we investigated the resistance reaction of lines with or without these genes to BaYMV-I and -III. All the pyramiding lines showed complete resistance to BaYMV-I and -III. This study anticipated the resistance to BaYMV-III of Mokusekko 3 by the multiple effect of the rym1 and rym5 genes.