In South American natural grasslands coexist species of C4 grasses with a higher growth strategies and species with low growth strategies. Biological nitrogen fixation (BNF) is very important for these grasses because the soil is nutrient poor. The understanding of this process may help explain the vegetation dynamics and improve the management of these grasslands. The objective of this work was to evaluate the association of four native grasses with three genus of diazotrophic bacteria under two levels of fertilization, and to determine the contribution of BNF. The four most abundant C4 grasses of South America natural grasslands (Axonopus affinis and Paspalum notatum, high growth strategies; Andropogon lateralis and Aristida laevis, low growth strategies) were cultivated in a greenhouse in a randomized block design with four replications. In a first experiment, two fertilization treatments were tested: 50mgPkg−1 soil+100mgNkg−1 soil (N+P) and a control (without N and P). The number of diazotrophic bacteria for the Azotobacter, Azospirillum and Herbaspirillum genus in the rhizosphere, rhizoplane and endorhizoplane were quantified. In a second experiment, BNF contribution was determined through natural 15N abundance technique. The results of this study point out that the genus of bacteria Azotobacter, Azospirillum and Herbaspirillum colonize the root system of these native grasses, contributing to the nitrogen nutrition of these species. Colonization increased with time, but N and P fertilization reduced colonization. Our results also showed that the N-fixation potential differ among species with different growth strategies. Aristida laevis (low growth strategy species) is the grass with the greater BNF contribution. P. notatum (high growth strategy species) showed great ability to absorb N from soil, with low contribution of BNF, being species with great response to fertilization.