In order to contribute for the optimization of biological nitrogen fixation (BNF) associated with cowpea in Cerrado areas in the Northeast region of Brazil, this work aimed to analyze the diversity of rhizobial populations in eight areas of Cerrado, during a soybean and rice-cowpea rotation. Morphological traits (mucous production and colony morphology), genotypic analyzes (ARDRA 16S) and intrinsic resistance to antibiotics were determined for a collection of isolates captured using cowpea as a host-plant. The morphological data showed a inverse correlation (p < 0.05) between the number of legume (soybean and cowpea) crops, according to the history of each area, and rhizobium diversity, estimated by the Shannon-Weaver index. ARDRA data showed that native Cerrado areas were exclusively colonized by Bradyrhizobium elkanii, corroborating previous data. In the areas where legumes were grown, we observed two distinct situations: where soybean only were grown, a high proportion of B. japonicum was found, and where soybean and cowpea were grown, we observed more B. elkanii. The analysis of antibiotic resistance revealed five different profiles. High percentage of antibiotic resistant Bradyrhizobium spp. isolates were found in the areas cultivated for a long time, whereas the native area and areas with a few crops had fewer resistant strain. There was an inverse relationship between intrinsic antibiotic resistance and rhizobial diversity, while the last decreases as more legume crops are introduced into the area, the former increases, suggesting that the presence of legumes may provide ecological conditions to select specific rhizobium groups, which acquire competitiveness traits and become successfully established.