Soil flooding or waterlogging is a major abiotic stress in upland crops. In barley, there have been several reported studies of selection for flooding-tolerant genotypes, but it is difficult to obtain varietal rankings that are consistent among researchers. Our objectives were to establish experimental conditions that could be applied by other research groups and to verify the varietal ranking conducted in an earlier study. We conducted greenhouse experiments on 14 barley varieties. At the 2.5-leaf stage, they were flooded with 0% or 0.1% soluble starch solution (mimicking reducing conditions). At 13 to 15 days after the start of treatment, the degree of leaf injury and the shoot dry weight ratio (treatment:control) were recorded. Reliable and highly repeatable results were obtained for the criterion of leaf injury under reducing conditions, whereas shoot dry weight ratio was unstable. The varieties OUJ820 and OUA301 were highly tolerant, whereas OUA002 and OUJ247 were sensitive; these results matched those of the earlier study. The experimental conditions that we developed here may be useful for selection testing and genetic analysis of flooding tolerance in other laboratories.