Aluminum (Al) toxicity is one of the most widespread problems of ion toxicity stress in plants and is a major limiting factor to crop productivity in an acid soil. To select Al tolerant plants, a quick, efficient screening method is needed. Our objective is to compare two Al tolerance screening methods for Medicago truncatula. In the first method, six accessions of M. truncatula seedlings were germinated for 24 h, then grown in an agarose medium, containing 5 levels of Al. Root length was measured at 24, 48 and 72 h after transfer. Relative root lengths (RRL, defined as root length at a particular Al level divided by that at no Al) differed among accessions particularly after 48 h. In the second method, seedlings were exposed to Al at the same levels as the first experiment for 12 h, then transferred to Al-free agarose medium, and root regrowth measured. The RRL during root regrowth differed significantly among cultivars. Based on root elongation in an Al- containing medium, A17 had the highest RRL, whereas PI 577609 was the most Al-tolerant in the root regrowth method. PI 535614 was the most Al sensitive in both experiments. Thus, ranking of Al-tolerance of M. truncatula cultivars differed depending on the Al-toxicity screening system. In the future, M. truncatula cultivars will be screened in an Al-toxic soil and results compared with these short-term screening methods to determine whether either of these methods is suitable for identifying Al-tolerant cultivars.