Azospirillum is a rhizobacterial genus containing plant growth-promoting species associated with different crops worldwide. Azospirillum brasilense strains exhibit a growth-promoting effect by means of phytohormone production and possibly by N2 fixation. However, one of the most important factors for increase in crop yield by plant growth-promoting rhizobacteria is the survival of the inoculant in the rhizosphere, which is not always achieved. The objective of this study was to develop quantitative PCR protocols for strain-specific quantification of A. brasilense FP2. A novel approach was applied to identify strain-specific DNA sequences based on comparison of genomic sequences within the same species. The draft-genome sequence of A. brasilense FP2 and Sp245 were aligned, FP2-specific regions were filtered and checked for other possible matches in public databases, Strain-specific regions were then selected to design and evaluate strain-specific primer pairs. The primer pairs AzoR2.1, AzoR2.2, AzoR5.1, AzoR5.2, and AzoR5.3 were strain-specific for A. brasilense FP2. These primer pairs were used to monitor quantitatively the population of A. brasilense in wheat roots under sterile and non-sterile growth conditions. In addition, co-inoculations with other plant-growth promoting bacteria in wheat were performed under non-sterile conditions. Results showed that A. brasilense FP2 inoculated to wheat roots is highly competitive and achieve high cell numbers (∼10(7) CFU/g of fresh weight root) in the rhizosphere even under non-sterile conditions and when co-inoculated with other rhizobacteria, maintaining the population rather stable for at least up to 13 days after inoculation. The strategy used here can be applied for other organisms whose genome sequences are available.