Soil salinity is a major constraint for the global agricultural production. For many decades, Na+ exclusion from uptake has been the key trait targeted in breeding programs; yet, no major breakthrough in creating salt-tolerant germplasm was achieved. In this work, we have combined the microelectrode ion flux estimation (MIFE) technique for non-invasive ion flux measurements with confocal fluorescence dye imaging technique to screen 45 accessions of barley to reveal the relative contribution of Na+ exclusion from the cytosol to the apoplast and its vacuolar sequestration in the root apex, for the overall salinity stress tolerance. We show that Na+/H+ antiporter-mediated Na+ extrusion from the root plays a minor role in the overall salt tolerance in barley. At the same time, a strong and positive correlation was found between root vacuolar Na+ sequestration ability and the overall salt tolerance. The inability of salt-sensitive genotypes to sequester Na+ in root vacuoles was in contrast to significantly higher expression levels of both HvNHX1 tonoplast Na+/H+ antiporters and HvVP1 H+-pumps compared with tolerant genotypes. These data are interpreted as a failure of sensitive varieties to prevent Na+ back-leak into the cytosol and existence of a futile Na+ cycle at the tonoplast. Taken together, our results demonstrated that root vacuolar Na+ sequestration but not exclusion from uptake played the main role in barley salinity tolerance, and suggested that the focus of the breeding programs should be shifted from targeting genes mediating Na+ exclusion from uptake by roots to more efficient root vacuolar Na+ sequestration.