In addition to drought and extreme temperatures, soil salinity represents a growing threat to crop productivity. Among the cereal crops, barley is considered as notably salt tolerant, and cultivars show considerable variation for tolerance towards salinity stress. In order to unravel the molecular mechanisms underlying salt stress tolerance and to utilize the natural genetic variation of barley accessions, a series of hydroponics-based salinity stress experiments was conducted using two genetic mapping parents, cvs Steptoe and Morex, which display contrasting levels of salinity tolerance. The proteome of roots from both genotypes was investigated as displayed by two-dimensional gel electrophoresis, and comparisons were made between plants grown under non-saline and saline conditions. Multivariate analysis of the resulting protein patterns revealed cultivar-specific and salt stress-responsive protein expression. Mass spectrometry-based identification was successful for 26 out of 39 selected protein spots. Hierarchical clustering was applied to detect similar protein expression patterns. Among those, two proteins involved in the glutathione-based detoxification of reactive oxygen species (ROS) were more abundant in the tolerant genotype, while proteins involved in iron uptake were expressed at a higher level in the sensitive one. This study emphasizes the role of proteins involved in ROS detoxification during salinity stress, and identified potential candidates for increasing salt tolerance in barley.