Chloride (Cl-) is an essential micronutrient for plant growth, but can be toxic at high concentrations resulting in reduced growth and yield. Although saline soils are generally dominated by both sodium (Na+) and Cl- ions, compared to Na+ toxicity, very little is known about physiological and genetic control mechanisms of tolerance to Cl- toxicity. In hydroponics and field studies, a bread wheat mapping population was tested to examine the relationships between physiological traits [Na+, potassium (K+) and Cl- concentration] involved in salinity tolerance (ST) and seedling growth or grain yield, and to elucidate the genetic control mechanism of plant Cl- accumulation using a quantitative trait loci (QTL) analysis approach. Plant Na+ or Cl- concentration were moderately correlated (genetically) with seedling biomass in hydroponics, but showed no correlations with grain yield in the field, indicating little value in selecting for ion concentration to improve ST. In accordance with phenotypic responses, QTL controlling Cl- accumulation differed entirely between hydroponics and field locations, and few were detected in two or more environments, demonstrating substantial QTL-by-environment interactions. The presence of several QTL for Cl- concentration indicated that uptake and accumulation was a polygenic trait. A major Cl- concentration QTL (5A; barc56/gwm186) was identified in three field environments, and accounted for 27-32% of the total genetic variance. Alignment between the 5A QTL interval and its corresponding physical genome regions in wheat and other grasses has enabled the search for candidate genes involved in Cl- transport, which is discussed.