Ion homeostasis is considered to be one of the most important mechanisms underlying salt stress tolerance. We used the Steptoe × Morex barley doubled haploid population to screen for genetic variation in response to salinity stress at an early development stage in a hydroponics system, focusing on ion homeostasis. Salinity induced a strong adverse effect on growth of the parents and their derived population, with Steptoe as the more tolerant parent. Steptoe maintained higher concentrations of K⁺, Na⁺ and Cl⁻ in the roots and a similar shoot/root ion ratio (<1) under stress conditions compared to control conditions. In contrast, Morex had higher concentrations of these ions in the shoots under stress and a doubled shoot/root ion ratio relative to control conditions, indicating that salt exclusion might contribute to the higher tolerance of Steptoe. Correlation and path analysis demonstrated that shoot Cl⁻ contents most strongly affected salt tolerance and suggest that both Na⁺ and Cl⁻ contents are important for salinity stress tolerance in barley. We identified 11 chromosomal regions involved in the control of the variation observed for salt tolerance and various salt stress response traits, including Na⁺, Cl⁻ and K⁺ contents in shoots. Two specific regions on chromosomes 2H and 3H were found controlling ion contents and salt tolerance, pointing to genes involved in ion homeostasis that contribute to salt tolerance.