Burkholderia species are extremely multidrug resistant, environmental bacteria with extraordinary bioremediation and biocontrol properties. At the same time, these bacteria cause serious opportunistic infections in vulnerable patient populations while some species can potentially be used as bioweapons. The complete DNA sequence of more than 10 Burkholderia genomes provides an opportunity to apply functional genomics to a collection of widely adaptable environmental bacteria thriving in diverse niches and establishing both symbiotic and pathogenic associations with many different organisms. However, extreme multidrug resistance hampers genetic manipulations in Burkholderia. We have developed and evaluated a mutagenesis system based on the homing endonuclease I-SceI to construct targeted, non-polar unmarked gene deletions in Burkholderia. Using the cystic fibrosis pathogen Burkholderia cenocepacia K56-2 as a model strain, we demonstrate this system allows for clean deletions of one or more genes within an operon and also the introduction of multiple deletions in the same strain. We anticipate this tool will have widespread environmental and biomedical applications, facilitating functional genomic studies and construction of safe strains for bioremediation and biocontrol, as well as clinical applications such as live vaccines for Burkholderia and other Gram-negative bacterial species.