Aluminum (Al) toxicity and drought are two major constraints on plant growth in acidic soils, negatively affecting crop performance and yield. Genotypic differences in the effects of Al/low pH and polyethyleneglycol (PEG) induced drought stress, applied either individually or in combination, were studied in Tibetan wild (XZ5, drought-tolerant; XZ29, Al-tolerant) and cultivated barley (Al-tolerant Dayton; drought-tolerant Tadmor). Tibetan wild barley XZ5 and XZ29 had significantly higher H+-ATPase, Ca2+Mg2+-ATPase, and Na+K+-ATPase activities at pH 4.0+Al+PEG than Dayton and Tadmor. Moreover, XZ5 and XZ29 possessed increased levels in reduced ascorbate and glutathione under these conditions, and antioxidant enzyme activities were largely stimulated by exposure to pH 4.0+PEG, pH 4.0+Al, and pH 4.0+Al+PEG, compared to a control and to Dayton and Tadmor. The activity of methylglyoxal (MG) was negatively correlated with increased levels of glyoxalase (Gly) I and Gly II in wild barley. Microscopic imaging of each genotype revealed DNA damage and obvious ultrastructural alterations in leaf cells treated with drought or Al alone, and combined pH 4.0+Al+PEG stress; however, XZ29 and XZ5 were less affected than Dayton and Tadmor. Collectively, the authors findings indicated that the higher tolerance of the wild barley to combined pH 4.0+Al+PEG stress is associated with improved ATPase activities, increased glyoxalase activities, reduced MG, and lower reactive oxygen species levels (like O2− and H2O2) due to increased antioxidant enzyme activities. These results offer a broad comprehension of the mechanisms implicated in barley’s tolerance to the combined stress of Al/low pH and drought, and may provide novel insights into the potential utilization of genetic resources, thereby facilitating the development of barley varieties tolerant to drought and Al/low pH stress.