This study analysed the resistance and susceptibility of several barley cultivars harbouring one of six resistance genes (rym1–6) to Japanese Barley yellow mosaic virus (BaYMV) isolate JK05 using infectious RNA transcripts. At the cellular level, the virus replicated efficiently in mesophyll protoplasts from rym3 cultivars, less efficiently in protoplasts from a rym2 cultivar and poorly or not at all in protoplasts from other rym cultivars. None of the rym cultivars was susceptible at the whole-plant level. These results indicate that rym2 and rym3 cultivars are resistant to this virus isolate at the movement level, whereas the other rym cultivars are resistant at the cellular level. In addition, among five barley cultivars without known resistance genes, it was found that the cv. Haruna Nijo was resistant to this virus isolate at the cellular level. Based on the host responses, it was inferred that the BaYMV isolate JK05 is in the Japanese pathotype II group. A possible mechanism for how resistance-breaking variants continually emerge in fields is discussed.