Aluminum (Al) toxicity in acid soils is one of the major limitations to alfalfa (Medicago sativa subsp. sativa L.) production in the USA. Developing Al tolerant alfalfa cultivars is an alternative to overcome this major limitation. Diploid non-cultivated species of alfalfa (M. sativa subsp. coerulea) were found to be an important reservoir of useful genes for tolerance to Al. Previously, two genomic regions associated with Al tolerance were identified in this diploid species using RFLP markers and single marker analysis in conjunction with callus growth bioassay. This study is being conducted to flank the previously identified Al tolerance QTL with simple sequence repeat (SSR) molecular markers to be used for marker-assisted introgression of the QTL into cultivated tetraploid alfalfa. A genetic linkage map is being constructed using SSR markers on a population of 130 BC₁ lines derived from the cross between an Al-sensitive genotype and an Al-tolerant genotype. So far one major QTL was established to be located on chromosome 1 which explained 30% of the phenotypic variation. The marker alleles significantly associated with this Al tolerance QTL and their effects on Al tolerance were examined. The SSR markers flanking this QTL can be used in marker-assisted selection and pyramiding this QTL to enhance Al tolerance.