<i>Aims</i> Phosphorus (P) is usually stratified in the topsoil layer under pasture, due to the broadcast application of fertiliser, excreta and leaf-litter deposition on the soil surface, and minimal soil disturbance. The objective of this study was to investigate root proliferation and P acquisition in response to P stratification by comparing two <i>Trifolium subterraneum</i> cultivars with contrasting root morphologies.<br/><i>Methods</i> Clover micro-swards were grown with deficient, constrained and sufficient P supplied in a topsoil layer overlying a P-deficient subsoil that mimicked the stratification of P that occurs under pasture. Phosphorus labelled with ³³P- and ³²P-radioisotope tracer was mixed throughout the topsoil and subsoil layers, respectively.<br/><i>Results</i> The shoot yield and total plant P uptake of the cultivars increased in response to increased topsoil P supply. The length of roots produced by the cultivars was equivalent in each of the P treatments, although the specific root length achieved by the cultivars was substantially different. In the P-constrained and P-sufficient treatments, ~91% and ~ 99% of total plant P was acquired by topsoil roots, respectively. In contrast, subsoil roots acquired 60-74% of total plant P in the P-deficient treatment.<br/><i>Conclusions</i> Topsoil roots were most important for P acquisition when P was highly stratified, whereas subsoil roots contributed to P acquisition when P was uniformly distributed throughout the P-deficient soil profile. Selection for prolific nutrient-foraging roots, in conjunction with plasticity for subsoil exploration, may improve the P-acquisition efficiency of <i>T. subterraneum</i> genotypes and confer adaptability across a range of soil-P environments.