Technical Abstract: Increased soil Aluminum (Al3+) toxicity has resulted in significant losses in wheat (Triticum aestivum L.) yield with a gradual decline in soil pH in many regions of the world. Elucidating the expression patterns of Al-regulated wheat genes occurring under Al stress may lead to discovery of new genes and further understanding the molecular mechanisms of wheat tolerance to Al toxicity. To enrich differentially expressed sequence tags (ESTs) for Al tolerance, cDNA subtraction libraries were generated from Al-stressed roots of two wheat near-isogenic lines (NILs) contrasting in Al-tolerance gene(s) from the Al-tolerant cultivar Atlas 66, using suppression subtractive hybridization (SSH). Expression patterns of the ESTs were investigated with nylon filter arrays containing 614 cDNA clones from the subtraction library. Gene expression profiles indicated that 72 ESTs were up-regulated in the tolerant NIL in response to Al stress. These highly expressed ESTs showed high homology with genes involved in signal transduction, oxidative stress alleviation and membrane structure, transportation and other functions. Under Al stress, the Al-tolerant NIL may posses altered structure and function of the cell wall, plasma membrane, and mitochondrion. The wheat response to Al stress may involve complicated defense-related signaling and metabolic pathways, but genes related to malate exudation, an important physiological mechanism of Al tolerance, were not identified in this study.