ABSTRACT The genotypic diversity of antibiotic-producing Pseudomonas spp. provides an enormous resource for identifying strains that are highly rhizosphere competent and superior for biological control of plant diseases. In this study, a simple and rapid method was developed to determine the presence and genotypic diversity of 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas strains in rhizosphere samples. Denaturing gradient gel electrophoresis (DGGE) of 350-bp fragments of phlD , a key gene involved in DAPG biosynthesis, allowed discrimination between genotypically different phlD ⁺ reference strains and indigenous isolates. DGGE analysis of the phlD fragments provided a level of discrimination between phlD ⁺ genotypes that was higher than the level obtained by currently used techniques and enabled detection of specific phlD ⁺ genotypes directly in rhizosphere samples with a detection limit of approximately 5 × 10 ³ CFU/g of root. DGGE also allowed simultaneous detection of multiple phlD ⁺ genotypes present in mixtures in rhizosphere samples. DGGE analysis of 184 indigenous phlD ⁺ isolates obtained from the rhizospheres of wheat, sugar beet, and potato plants resulted in the identification of seven phlD ⁺ genotypes, five of which were not described previously based on sequence and phylogenetic analyses. Subsequent bioassays demonstrated that eight genotypically different phlD ⁺ genotypes differed substantially in the ability to colonize the rhizosphere of sugar beet seedlings. Collectively, these results demonstrated that DGGE analysis of the phlD gene allows identification of new genotypic groups of specific antibiotic-producing Pseudomonas with different abilities to colonize the rhizosphere of sugar beet seedlings.