Within the semi-arid region of south-eastern Australia, high levels of subsoil boron (B) in alkaline soil can limit production of dryland crops. The aim of this research was to investigate the whole plant response to a range of subsoil-extractable B concentrations for a number of crop and weed species common to agricultural areas of South Australia. Specifically, the objectives were to determine (a) the morphological response of the entire root system to high subsoil B and (b) the available B concentrations in subsoil critical for expression of shoot traits commonly used in selection of B tolerance. Barley grass (Hordeum glaucum L.), crop barley (Hordeum vulgare) variety Clipper and breeders’ line VB9953, fababean (Vicia faba var. Fjiord), Lincoln weed (Diplotaxis tenuifolia L.), prickly lettuce (Lactuca serriola), and evening primrose (Oenothera stricta L.) were grown in sealed PVC cylinders (500 mm deep by 150 mm diam.) containing a sandy soil. The concentration of extractable B in the topsoil (0–0.20 m), considered non-toxic, was 0.5 mg/kg for all cylinders but a range of B treatments (0.5, 2.4, 4.3, 6.8, or 12.2 mg/kg) was applied directly to the subsoil (0.30–0.50 m). Increasing the concentration of extractable B in the subsoil decreased root dry weight in this region, but did not reduce water use from subsoil by barley grass or evening primrose. The response of the roots in the topsoil and subsequent responses in the shoot also differed among species. Symptoms of B toxicity in shoots of all the species were observed at subsoil-extractable B concentrations of 12.2 mg/kg and at lower concentrations in some of the crop and weed species. Shoot growth, total water use, and root growth in topsoil of Clipper and Lincoln weed were severely impaired by high subsoil-extractable B, as was topsoil root growth in evening primrose, with the reduction in the weed species being mostly associated with a decrease in taproot dry weight. Barley grass, VB9953, evening primrose, and to a lesser extent fababean and prickly lettuce, maintained shoot growth at all subsoil-extractable B concentrations, despite a reduction in subsoil water use by VB9953. Prickly lettuce and VB9953 also sustained root growth in the topsoil whilst fababean and barley grass increased root growth in the topsoil in response to high subsoil extractable B. There was no direct relationship between the quantity of B accumulated in shoots and detrimental effects on growth. Furthermore, there appeared to be no direct relationship between water uptake and B uptake since irrespective of the effect of subsoil B on either subsoil or total water use, shoot B concentration increased in all the species/genotypes as subsoil B increased. The degree to which plants were deemed to exhibit tolerance was, therefore, highly dependent upon the trait used for assessment. One suggestion in the current study is that shoot dry matter in B toxic soil can be a consistent parameter for considering varieties for tolerance to B toxicity.