Trifolium subterraneum L. is widely grown in the phosphorus (P) deficient soils of southern Australia. However, this pasture legume has a high critical external P requirement and requires frequent applications of P fertiliser to achieve high productivity. Twenty-six genotypes of T. subterraneum were grown to determine: (i) differences in shoot growth and P acquisition under low-P supply; (ii) the root morphological traits important for P acquisition; and (iii) the feasibility of selection among genotypes for these root morphological traits. Micro-swards of each genotype were grown with a topsoil layer that was either moderately P-deficient or had P supplied in excess of the critical requirement for maximum yield; the subsoil layer was P-deficient. Yield and P content of shoots and roots were determined after 5 weeks’ growth, and root samples were assessed for diameter, length and root hair length. All genotypes were equally highly productive when excess P was supplied. However, relative shoot yield in the moderately P-deficient soil ranged from 38–71%. Total root length ranged from 63–129 m pot–1, and was correlated with total plant P uptake (R2 = 0.78, P < 0.001). Variation was also observed in average root diameter (0.29–0.36 mm) and root hair length (0.19–0.33 mm). These traits were combined with root length to calculate the total surface area of the root hair cylinder, which was also correlated with total plant P uptake (R2 = 0.69, P < 0.001). The results demonstrated that there was significant variation in P acquisition efficiency and shoot yield among genotypes of T. subterraneum when grown in P-deficient soil, and that root length was important for improved P uptake. The results indicate potential to identify superior genotypes that achieve improved P acquisition and higher shoot yields in low-P soil.