Denaturing gradient gel electrophoresis (DGGE) and SYBR Green I quantitative real-time PCR (qPCR) approaches were used to assess respectively the molecular diversity and the quantity of the nifH gene sequences in the rhizospheres of two cultivars of sorghum sown in Cerrado soil with contrasting levels of nitrogen fertilizer. DGGE fingerprinting showed that for both cultivars the presumptive nitrogen-fixing populations in the rhizospheres were more diverse than in bulk soil. Sequencing of nifH gene fragments obtained from DGGE bands revealed that members of the order Rhizobiales were prevalent among the dominant diazotrophs. Discriminant analysis of DGGE profiles resulted into three groups formed by (i) cultivar BRS 308 sown with high level of nitrogen, (ii) cultivar BRS 308 sown with low level of nitrogen and cultivar BRS 310 sown either with low or high levels of nitrogen and (iii) bulk soil, showing that the nitrogen fertilization influenced the nifH gene sequence diversity only in the rhizosphere of cultivar BRS 308. When the quantity of the nifH gene sequences was determined by q-PCR, 2.4 × 105 to 1.3 × 107 copies/g of soil were detected. The highest number of nifH gene copies was observed in the rhizosphere of cultivar BRS 308 treated with low amount of fertilizer, and a reduction in nifH density was observed in the rhizospheres of both sorghum cultivars when high levels of nitrogen were applied. Thus, both the amount of nitrogen fertilizer and the cultivar are important factors influencing the structure and amount of diazotrophs in sorghum.