Phosphorus (P) is usually concentrated in the uppermost layers of the soil profile under pasture, hence topsoil root allocation is important for maximising P acquisition. However, total root length was recently found to be a marginally better predictor of variation in P uptake among twenty-six genotypes of subterranean clover (<i>Trifolium subterraneum</i> L.) when compared to topsoil root length alone. This result prompted a preliminary assessment of P acquisition by subsoil roots. Micro-swards of two cultivars were grown with a topsoil layer that was either P-deficient or amended with P for improved plant growth, overlying a low-P subsoil that contained ³²P-labelled phosphate. Both cultivars produced less shoot dry mass under P constraint, and the cultivar that allocated more root length to the subsoil layer produced a larger shoot dry mass in the P-deficient soil. This cultivar also recovered more ³²P-labelled phosphate from the subsoil layer in both P treatments. Therefore, variation exists for subsoil P acquisition and this trait may be important for determining shoot yield in P-deficient soil.