The objective of this study was to quantify the genetic variability and determine the inheritance of Al tolerance in lucerne (or alfalfa) using a 4-parent diallel mating design. Regrowth root length (RRL), along with root length (RL) and total root length (TRL), was measured to indicate relative Al stress response using a hydroponic system in a 3 lM Al solution at pH 4.5. A diallel analysis indicated the significance of general combining ability (gca) variance for RRL but not the specific combining ability (sca) variance; the same result was obtained for TRL but not for RL. For both RRL and TRL, genetic variance appeared to be more important than the environmental variance. For RRL, a strong but non-significant correlation was indicated between parental performance and their gca effects; while mid-parent heterosis and/or over-dominance were detected, as associated with the expressed sca effects in several combinations. These results suggest the complex genetic nature and expression of Al tolerance in the 4-parent diallel crossing system tested. Al-tolerant parent, GAAT'S', was the most promising parent, conferring the highest gca effects for RRL as well as for other two characters. The existence of significant gca variance in RRL may also suggest the feasibility of improving Al tolerance through enhanced root regrowth using phenotypic recurrent mass selection to pyramid desirable Al-tolerant genes, focussing on parental lines and/or elite individual plants expressing long regrowth roots.