A soil-based screening method was used to test tolerance of wheat genotypes to acidity. Plants were grown for 6 days in an acid soil with the pH adjusted to 3.9–5.8. The number and length of roots were measured. To validate the method, 12 wheat cultivars of known acidity tolerance and one acid-sensitive barley cultivar were grown on an acid soil in the field with or without amelioration of subsoil acidity. The relative yields of these wheat genotypes on the soil with subsoil acidity ranged from 50 to 89% of yields on soil without subsoil acidity, and were correlated with root growth parameters obtained in the glasshouse. The best correlation was obtained between relative yields in the field (y) and root length per plant (x) at pH 3.9 in the glasshouse (y = –43 + 52*log x, r = 0.95) or root length per plant at pH 3.9 as a percentage of root length at pH 4.8 (y = 1.2 + 46*log x, r = 0.94). Following validation of the glasshouse screening method in the field, 115 wheat genotypes, including cultivars and breeding lines, were screened in the glasshouse. A substantial genotypic variation in acidity tolerance was found, with root length per plant at pH 3.9 ranging from 66 to > 350 mm. Many Western Australian breeding lines displayed better tolerance than existing tolerant wheat cultivars. The screening system can be instrumental in breeding wheat for increased tolerance to acid soils.