Technical Abstract: To understand the mechanisms of aluminum (Al) tolerance and identify genes responsible for Al tolerance in wheat (Triticum aestivum L.), suppression subtractive hybridization (SSH) libraries were constructed from Al-stressed roots for two wheat near- isogenic lines (NILs), Chisholm-T (Al-tolerant) and Chisholm-S (Al-sensitive). A total of 1628 cDNA clones representing 1065 putative genes from the libraries were spotted on glass slides. Relative expression levels of those genes were compared between Chisholm-T and Chisholm-S at seven time points of Al stress: 15 min, 1 h, 3 h, 6 h, 1 d, 3 d and 7 d. Fifty-seven genes were differentially expressed for at least one time point of Al treatment. Among them, 28 genes including genes for aluminum-activated malate transporter-1, ent-kaurenoic acid oxidase-1, B-glucosidase, lectin, histidine kinase, and phospoenolpyruvate carboxylase showed more abundant transcripts in Chisholm-T and therefore may facilitate Al tolerance. In addition, a set of genes related to senescence and starvation of nitrogen, iron and sulfur, such as copper chaperone homolog, nitrogen regulatory gene-2, yellow stripe-1, and methylthioribose kinase, was highly expressed in Chisholm-S under Al stress. The results suggest that Al tolerance may be co-regulated by multiple genes with diverse functions, and those genes abundantly expressed in Chisholm-T may play important roles in enhancing Al tolerance and protecting root growth under Al stress. The highly expressed genes in Chisholm-S under Al stress may be symptomatic of root growth repression and restricted uptake of essential nutrient elements, leading to root senescence.