ABSTRACT Fluorescent Pseudomonas spp. that produce 2,4-diacetylphloroglucinol (2,4-DAPG) have biocontrol activity against damping-off, root rot, and wilt diseases caused by soilborne fungal pathogens, and play a key role in the natural suppression of Gaeumannomyces graminis var. tritici, known as take-all decline. Diversity within phlD, an essential gene in the biosynthesis of 2,4-DAPG, was studied by restriction fragment length polymorphism (RFLP) analysis of 123 2,4-DAPG-producing isolates from six states in the United States and six other locations worldwide. Clusters defined by RFLP analysis of phlD correlated closely with clusters defined previously by BOX-polymerase chain reaction (PCR) genomic fingerprinting, indicating the usefulness of phlD as a marker of genetic diversity and population structure among 2,4-DAPG producers. Genotypes defined by RFLP analysis of phlD were conserved among isolates from the same site and cropping history. Random amplified polymorphic DNA analyses of genomic DNA revealed a higher degree of polymorphism than RFLP and BOX-PCR analyses. Genotypic diversity in a subset of 30 strains representing all the phlD RFLP groups did not correlate with production in vitro of monoacetylphloroglucinol, 2,4-DAPG, or total phloroglucinol compounds. Twenty-seven of the 30 representative strains lacked pyrrolnitrin and pyoluteorin biosynthetic genes as determined by the use of specific primers and probes.