Aluminum (Al) toxicity is a major limitation for wheat production in acidic soils. Al tolerance gene on chromosome 4DL originated from Brazilian wheat has been extensively studied and can provide partial protection from Al damage. To identify new sources of Al tolerance for enhancement of Al tolerance of wheat cultivars, 430 wheat accessions including elite wheat breeding lines from USA and other American countries, landraces and commercial cultivars from East Asia and synthetic Wheat lines from CYMMIT were screened for Al tolerance by evaluating relative root elongation under Al stress in hydroponics and by hematoxylin stain. Result showed that about 60 lines were highly tolerant to Al stress in hydroponics. These selected lines were subjected to screening with microsatellite markers linked to the Al tolerance QTL on 4DL and with the gene marker of Al-activated malate transporter (ALMT1). Among these 60 Al tolerant lines, many accessions from East Asia do not have the same ALMT1 allele as that in Atlas 66, although they had similar Al-tolerant phenotypes. This result suggests that ALMT1 may not be the only gene to control Al tolerance and Asian Al-tolerant accession may be different sources of Al tolerance from that of Brazilian wheat.