The acid, aluminum (Al) toxic soils found throughout the USA are a major limitation to the productivity of cultivated alfalfa (Medicago sativa ssp. sativa L.). One strategy to overcome this limitation is to develop Al tolerant alfalfa cultivars. The objective of this study was to identify quantitative trait loci (QTL) controlling Al tolerance in diploid Medicago sativa ssp. coerulea genotypes to be used for introgression of the QTL into cultivated, tetraploid alfalfa. RFLP markers were used in conjunction with a callus growth bioassay to identify Al tolerance QTL in an F₂ population, and confirm them in a backcross population. Single marker analysis was used to find significant (p<0.05) associations between RFLP markers and callus weight means. Three RFLP markers, UGAc191, UGAc471, and UGAc502 were associated with Al tolerance in the F₂ and backcross callus assays. A preliminary soil-based study was conducted with selected F₂ individuals to verify that QTL markers identified in tissue culture were also associated with Al tolerance in whole plants growing in soil. Accumulation of positive alleles and exclusion of negative alleles for the QTL markers identified in tissue culture resulted in higher Al tolerance in this preliminary soil-based assay. Finally, as a first step in introgressing these QTL into cultivated, tetraploid alfalfa, 2X-4X crosses were made between diploid F₁ hybrids and tetraploid genotypes of "CUF 101’, and five tetraploid progeny were obtained via 2n gametes. Marker alleles associated with Al tolerance were identified in all five progeny. These RFLP markers can be used to further pinpoint the location of Al tolerance QTL, and to introgress these QTL into cultivated, tetraploid alfalfa.