Root morphology and nutrient uptake processes are essential for acquisition of mineral resources from soil. However, our understanding of how root form and function have diverged across environments is limited. In this study, we addressed hypotheses of adaptive differentiation using three pairs of Helianthus species chosen as phylogenetically-independent contrasts with respect to native soil nutrients. Under controlled environmental conditions, root morphology, allocation, and nitrogen (N) uptake (using a 15N tracer) were assessed for seedlings under both high and low N treatments. Species native to low nutrient soils (LNS) had lower total root length than those native to high nutrient soils (HNS), reflecting the slower growth rates of species from less fertile environments. Contrary to expectations, species did not consistently differ in specific root length, root tissue density, or root system plasticity, and species native to LNS had lower root : total mass ratio and higher 15N uptake rates than species native to HNS. Overall, these evolutionary divergences provide support for adaptive differentiation among species, with repeated evolution of slow-growing root systems suited for low resource availability in LNS. However, species native to LNS maintain a high capacity for N uptake, potentially as a means of maximising nutrient acquisition from transient pulses.