Salinity is a major problem for viticulture. Accumulation of sodium ions in berries can result in wine that is unpalatable, and accumulation in shoots negatively impacts on yield. We have identified a high affinity potassium transporter (HKT) from grapevine roots that is selective for sodium and no other monovalent cations when expressed in Xenopus laevis oocytes. We have cloned and functionally characterised four allelic variants of this HKT from two heterozygous grapevine rootstocks that possess contrasting salt tolerance. When expressed in Saccharomyces cerevisiae, two allelic variants strongly inhibited growth on high sodium medium, while another two allelic variants caused weaker growth inhibition. Furthermore, in Xenopus oocytes, we observed differences in the electrophysiological properties of the allelic variants. In the progeny of a cross between the two grapevine rootstocks, individuals that were homozygous for the toxic HKT allelic variants contained less shoot sodium, heterozygous individuals showed intermediate shoot sodium accumulation, and individuals homozygous for the less toxic HKT allele were less able to exclude sodium from leaves. These results indicate a role of this protein in the natural variation in shoot sodium exclusion across grapevine varieties, and provide novel insights into the mechanism of salinity tolerance mediated by HKT proteins in plants.