Salinity is a major factor limiting crop productivity. Rice (Oryza sativa), a staple crop for the majority of the world, is highly sensitive to salinity stress. To discover novel sources of genetic variation for salt tolerance-related traits in rice, we screened 390 diverse accessions under 14 days of moderate (9 dS·m-1) salinity. In this study, shoot growth responses to moderate levels of salinity were independent of tissue Na+content. A significant difference in root Na+content was observed between the major subpopulations of rice, with indica accessions displaying higher root Na+and japonica accessions exhibiting lower root Na+content. The genetic basis of the observed variation in phenotypes was elucidated through genome-wide association (GWA). The strongest associations were identified for root Na+:K+ratio and root Na+content in a region spanning ~575 Kb on chromosome 4, named Root Na+Content 4 (RNC4). Two Na+transporters, HKT1;1 and HKT1;4 were identified as candidates for RNC4. Reduced expression of both HKT1;1 and HKT1;4 through RNA interference indicated that HKT1;1 regulates shoot and root Na+content, and is likely the causal gene underlying RNC4. Three non-synonymous mutations within HKT1;1 were present at higher frequency in the indica subpopulation. When expressed in Xenopus oocytes the indica-predominant isoform exhibited higher inward (negative) currents and a less negative voltage threshold of inward rectifying current activation compared to the japonica-predominant isoform. The introduction of a 4.5kb fragment containing the HKT1;1 promoter and CDS from an indica variety into a japonica background, resulted in a phenotype similar to the indica subpopulation, with higher root Na+and Na+:K+. This study provides evidence that HKT1;1 regulates root Na+content, and underlies the divergence in root Na+content between the two major subspecies in rice.