Four bacterial strains were isolated from enrichment cultures inoculated with soil from Bien Hoa military base in Vietnam contaminated with the herbicides 2,4-dichlorophenoxyacetate (2,4-D) and 2,4,5-trichlorophenoxyacetate (2,4,5-T). They were classified as Pseudomonas aeruginosa BT1 2.2, Sphingomonas histidinilytica BT1 5.2, Bordetella petrii BT1 9.2, and Achromobacter xylosoxidans BT1 10.2, respectively. All 4 of them were able to degrade 2,4-D and 2,4,5-T during cultivation, but only the last 3 species used them as sole sources of carbon and free energy. We obtained a comprehensive insight into their degradation pathways by genomic analysis of these strains. A gene cluster with tfdCDEF genes was found in A. xylosoxidans BT1 10.2. The gene organization along with the amino acid sequences of the gene products are almost identical to those in B. petrii DSM12804. The B. petrii BT1 9.2 strain that we isolated has a full complement of the tfdABCDEF genes. Surprisingly, the gene organization along with the amino acid sequences of the gene products are virtually identical to those of Cupriavidus pinatubonensis JMP134, referred to as type I tfd genes, and clearly different from those of A. xylosoxidans and B. petrii DSM12804. Altogether, our enrichment approach has successfully resulted in boosting 3 different types of proteobacterial species that are equipped with metabolic pathways to use the herbicides as sole sources of carbon and free energy. We hypothesize that some of the corresponding genetic potential may have been recruited in recent mating events between these species and other members of the β- and γ-proteobacteria.