Aims: A causal relationship between salinity and oxidative stress tolerance and a suitability of using root antioxidant activity as a biochemical marker for salinity tolerance in barley was investigated. Methods: Net ion fluxes were measured from the mature zone of excised roots of two barley varieties contrasting in their salinity tolerance using non-invasive MIFE technique in response to acute and prolonged salinity treatment. These changes were correlated with activity of major antioxidant enzymes; ascorbate peroxidase, catalase, and superoxide dismutase. Results: It was found that genotypic difference in salinity tolerance was largely independent of root integrity, and observed not only for short-term but also long-term NaCl exposures. Higher K+ retention ability (and, hence, salinity tolerance) positively correlated with oxidative stress tolerance. At the same time, antioxidant activities were constitutively higher in a sensitive but not tolerant variety, and no correlation was found between SOD activity and salinity tolerance index during large-scale screening. Conclusion: Although salinity tolerance in barley correlates with its oxidative stress tolerance, higher antioxidant activity at one particular time does not correlate with salinity tolerance and, as such, cannot be used as a biochemical marker in barley screening programs.