Aluminum toxicity in acidic soils represents a significant environmental stressor that affects yields worldwide and is only expected to worsen. Breeding resistant varieties remains the most viable solution; however, fast and robust procedures to determine cultivar viability must be developed and applied to promising genotypes. This study explored historical and modern Lithuanian-bred barley cultivars using morphometrical and biochemical markers for Al resistance and sequence and expression analyses of potential candidate genes. Morphometric seedling measurements (relative root length reduction -13.65 ± 0.33% ( < 0.001) and root tolerance index 0.86 ± 0.44 after 72 h at 8 mM Al stress) revealed the modern cv. 'Ema DS' to be the most Al resistant, while biochemical assays offered a poor distinction between the Al-resistant and sensitive cultivars. Thus, we determined that morphometric parameters were more effective in the early screening for barley Al resistance. The genetic screening of well-established Al resistance markers in the barley citrate transporter HvAACT1 revealed a mismatch between the observed barley phenotypes and genotypes. Further testing was conducted through expression analyses of and seven aquaporin family genes, which revealed a correlation between the best empirical performance in cv. 'Ema DS' and a high (2.02 fold change, < 0.05) expression, despite the lack of established genetic markers, as well as a stress-induced significant upregulation of aquaporin TIP4;1 (2.45 fold change, < 0.05), suggesting previously undiscovered regulatory mechanisms of external and internal detoxification influencing Al resistance in Lithuanian barley cultivars, as well as potential future candidates for Al-resistant barley breeding programs.