Acid soil is a worldwide problem to crop production. Selection and breeding of barley cultivars tolerant to acid soil is one of the most useful approaches to increase productivity on acid soils. To make the selection more efficient, an understanding of the genetic behaviour of acid soil tolerance in barley is needed. For this purpose, a 6 X 6 diallel analysis was conducted in barley from crosses of six varieties differing in acid soil tolerance. The parent varieties included two known tolerant (Brindabella and Honen), two newly identified tolerant (CXHKSL and YSM1) and two sensitive ones (Gairdner and Baudin). The newly developed screening facilities were used to evaluate the root growth of parents and their F2s in acid soils. Results showed that the variance of GCA (General Combining Ability) was highly significant (P < 0.01) while that of SCA (Special Combining Ability) was not significant, indicating that the acid soil tolerance was mainly influenced by additive genic action. Honen had greatest GCA, followed by Brindabella, YSM1 and CXHKSL. A lowest negative GCA was found in Gairdner. The GCA values of the parent varieties were also consistent with their tolerance performance. Further studies will be conducted on whether the tolerance of different tolerant varieties was controlled by same gene(s).