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To clone and sequence polymerase chain reaction (PCR)-amplified glnB and nifH genes of the nitrogen-fixing bacteria Burkholderia brasilensis strain M130, B. tropicalis strain PPe8 and B. kururiensis strain KP23.The glnB and nifH gene fragments were amplified by PCR using universal degenerated primers. A very high percentage of similarity for the nifH (100%) and glnB (96%) genes was observed between strains M130 and KP23. A similarity of 100% for the nifH gene was also observed between strains M130 and PPe8. However, the identity for the glnB gene was 98% and the similarity 88%. The phylogenetic tree of the nifH gene showed a very high degree of similarity to the 16S rDNA gene.The nitrogen-fixing bacteria of the Burkholderia genus formed a cluster separated from the other species of the genus mainly when the nifH rather than the glnB gene was used to construct the phylogenetic tree.Knowledge of the nifH and glnB gene sequences of B. brasilensis, B. tropicalis and B. kururiensis will support new studies on the diversity of these diazotrophs in natural environments.