ABSTRACT Pseudomonas fluorescens strains producing the antibiotic 2,4-diacetylphloroglucinol (DAPG) have biocontrol activity against a broad spectrum of root and seedling diseases. In this study, we determined the effect of genotype on the ability to isolate and quantify introduced 2,4-DAPG producers from the rhizosphere of wheat using three different methods: traditional dilution plating on selective media, colony hybridization followed by polymerase chain reaction (PCR), and phlD-specific PCR-based dilution endpoint assay. Regression analysis of the population densities of 10 2,4-DAPG-producing P. fluorescens, representing five genotypes, determined by the three different methods demonstrated that the relationship was linear (P < 0.001) and the techniques were very similar (i.e., slopes equal to 1.0). The phlD-specific PCR-based assay had a slightly lower limit of detection than the other two methods (log 3.3 versus log 4.0 CFU/g of fresh root weight). With the colony hybridization procedure, we observed that the phlD probe, derived from strain P. fluorescens Q8r1-96, hybridized more strongly to colonies of BOX-PCR genotypes D (strains W2-6, L5.1-96, Q8r1-96, and Q8r2-96) and K (strain F113) compared with strains of genotypes A (Pf-5 and CHA0), B (Q2-87), and L (1M1-96 and W4-4). Colony hybridization alone overestimated the actual densities of some strains, thus requiring an additional PCR step to obtain accurate estimates. In contrast, population densities estimated for three of the bacterial treatments (strains CHA0, W2-6, and Q8r2-96) with the PCR-based assay were significantly (P < 0.041) smaller by 7.6 to 9.2% and 6.4 to 9.4% than population densities detected by the dilution plating and colony hybridization techniques, respectively. In this paper, we discuss the relative advantages of the different methods for detecting 2,4-DAPG producers.