Chromium (Cr) is one of the most toxic metals, posing serious threats to crop productivity, environmental quality, and human health. Cultivars differ in their ability to tolerate heavy metals. Therefore, this study was performed to evaluate the response of different barley cultivars against Cr stress. The study contained different barley cultivars: Sultan-17, Haider-93, Jau-17, Pearl-21, and Jau-21 and Cr stress levels 20 and 40 mg kg-1 soil. The findings demonstrate a significant decline in barley growth, physiological, biochemical, yield and ionic responses under chromium stress. Among varieties, Haider-93 showed maximum decrease in plant height (33.1%), shoot fresh and dry weight (62.2%, 53.4%), root fresh and dry weight (53.6%, 57.7%) under Cr2 stress while Sultan-17 exhibited comparatively lower reduction in these parameters: plant height (19.8%), shoot fresh and dry weight (26.1%, 22.4%), root fresh and dry weight (26.4%, 21.9%) under level of Cr stress. Chromium stress also negatively affected physiological traits, with Haider-93 showing a significant decline in relative water content (47.9%) and chlorophyll a (55.3%), while Sultan-17 had lower reductions (21.8% and 18.5%, respectively) in under 40 mg kg-1 Cr stress. Sultan-17 exhibited a stronger antioxidant response, with a 23.1% increase in ascorbic acid content, compared to 17.7% in Haider-93. Moreover, Haider-93 showed higher levels of H₂O₂ and MDA production and yield reduction compared to Sultan-17. The results suggest that Sultan-17 showed the highest tolerance to Cr toxicity. These findings provide insights to develop tolerant crops from Cr contaminated soils.