The mini-Tn7 vectors are universally applicable in gram-negative bacteria and thereby facilitate the manipulation of many organisms for which few genetic systems are available. These vectors, when provided with only the Tn7 site-specific transposition machinery, insert site and orientation specifically in the bacterial chromosome at an attTn7 site downstream of the essential glmS gene. A few bacteria, including Burkholderia spp., contain multiple glmS genes and therefore several attTn7 sites. Here we provide a protocol for application of the mini-Tn7 system in B. mallei as an example of bacteria with multiple glmS sites. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into B. mallei by conjugation, followed by selection of insertion-containing strains; and last, PCR verification of mini-Tn7 insertions. B. mallei possesses two glmS genes on chromosome 1 and Tn7 transposes to both sites, although transposition to attTn7-1 associated with glmS1 occurs in more than 90% of the clones examined. Transposition is efficient and the whole procedure from start to verification of insertion events can be done in less than 5 d. This first chromosome integration system in B. mallei provides an important contribution to the genetic tools emerging for Burkholderia spp. Vectors are available for gene complementation and expression, and gene fusion analyses.