The natural grasslands in South America have soils with low phosphorus (P) availability (1.0 to 7.5 mg kg−1), possibly altering the absorption and accumulation of P in grasses. We evaluated the chemical fractionation of P in the leaves of the most important grasses present in these grasslands to better understand the mechanisms involved in the storage of P. The grasses studied were Axonopus affinis and Paspalum notatum (fast tissue cycling and high nutrient demand) and Andropogon lateralis and Aristida laevis (slow tissue cycling and low nutrient demand). They were grown in pots filled with an Ultisol with two levels of P: control, and addition of 50 mg P kg–1. The main P fractions were the inorganic soluble (44%) and P in RNA (26%). Addition of P increased the total P concentration, following the order A. affinis (140%) > P. notatum (116%) > A. lateralis (81%) > A. laevis (21%). In conclusion, the species A. affinis and P. notatum responded to P fertilization with high variation and accumulating P in less-structural chemical forms, such as inorganic P. The species A. lateralis and A. laevis showed low variation in the concentration of P forms, with higher P concentrations in structural forms.