A large-scale glasshouse trial, including nearly 70 barley cultivars (5300 plants in total), was conducted over 2 consecutive years to investigate plant physiological responses to salinity. In a parallel set of experiments, plant salt tolerance was assessed by non-invasive microelectrode measurements of net K⁺ flux from roots of 3-day-old seedlings of each cultivar after 1 h treatment in 80 mm NaCl as described in our previous publication (Chen et al. 2005). K⁺ flux from the root in response to NaCl treatment was highly (P < 0.001) inversely correlated with relative grain yield, shoot biomass, plant height, net CO₂ assimilation, survival rate and thousand-seed weight measured in glasshouse experiments after 4–5 months of salinity treatment. No significant correlation with relative germination rate or tillering was found. In general, 62 out of 69 cultivars followed an inverse relationship between K⁺ efflux and salt tolerance. In a few cultivars, however, high salt tolerance (measured as grain yield at harvest) was observed for plants showing only modest ability to retain K⁺ in the root cells. Tissue elemental analysis showed that these plants had a much better ability to prevent Na⁺ accumulation in plant leaves and, thus, to maintain a higher K⁺/Na⁺ ratio. Taken together, our results show that a plant’s ability to maintain high K⁺/Na⁺ ratio (either retention of K⁺ or preventing Na⁺ from accumulating in leaves) is a key feature for salt tolerance in barley.