Subsurface soil acidity coupled with high levels of toxic Al is a major limiting factor in wheat production in many areas of the world. This study examined the effect of subsurface soil acidity on the growth and yield of two near-isogenic wheat genotypes differing in Al tolerance at a single genetic locus in reconstructed soil columns. In one experiment, plants were grown in columns with limed topsoil and limed or acidic subsurface soils, and received water only to the subsurface soil at a late part of the growth period. While shoot dry weight, ear number and grain yield of Al-tolerant genotype (ET8) were not affected by subsurface soil acidity, liming subsurface soil increased shoot weight and grain yield of Al-sensitive genotype (ES8) by 60% and ear number by 32%. Similarly, root length density of ET8 was the same in the limed and acidic subsurface soils, while the root length density of ES8 in the acidic subsurface soil was only half of that in the limed subsurface soil. In another experiment, plants were grown with limed topsoil and acidic subsurface soil under two watering regimes. Both genotypes supplied with water throughout the soil column produced almost twice the dry weight of those receiving water only in the subsurface soil. The tolerant genotype ET8 had shoot biomass and grain yield one-third higher than ES8 when supplied with water throughout the whole column, and had yield 11% higher when receiving water in the subsurface soil only. The tolerant genotype ET8 produced more than five times the root length in the acidic subsurface soil compared to ES8. Irrespective of watering regime, the amount of water added to maintain field capacity of the soil was up to 2-fold higher under ET8 than under ES8. The results suggest that the genotypic variation in growth and yield of wheat grown with subsurface soil acidity results from the difference in root proliferation in the subsurface soil and hence in utilizing nutrient and water reserves in the subsurface soil layer.