Aluminum (Al) toxicity is a major constrain for wheat production in acidic soils and the use of Al tolerant cultivars is a cost-effective solution to improve wheat productivity in acidic soils. Quantitative trait loci (QTLs) for wheat Al tolerance were analyzed using simple sequence repeats (SSRs) in a population of 192 F6 recombinant inbred lines (RILs) derived from a cross between an Al-tolerant cultivar Atlas 66 and an Al-sensitive cultivar Chisholm. Al tolerance was evaluated by measuring relative root growth and root response to hematoxylin stain after Al treatment in hydroponics. After 992 SSR markers were screened for polymorphisms between the parents and bulks, two QTLs were identified. One major QTL was mapped on chromosome 4D that co-segregated with the Al activated malate transporter gene (ALMT1), and another minor QTL located on chromosome 3BL. These two QTLs together accounted for about 57% of the phenotypic variation for Al tolerance based on the hematoxylin staining score. The new minor QTL on 3BL has a significant effect on Al tolerance suggesting more than one QTL for Al tolerance in wheat cultivar Atlas 66. The two QTLs were also verified in other two populations with Atlas 66 as the Al tolerant parent. Several SSR markers closely linked to the QTLs were identified and have potential to be used for marker-assisted selection to improve Al tolerance of wheat cultivars in breeding programs.