The study of the minimum set of genes required to sustain life is a fun-damental question in biological research. Recent studies on bacterial essential genessuggested that between 350 and 700 genes are essential to support autonomousbacterial cell growth. Essential genes are of interest as potential new antimicrobialdrug targets; hence, our aim was to identify the essential genome of the cystic fibro-sis (CF) isolateBurkholderia cenocepaciaH111. Using a transposon sequencing (Tn-Seq) approach, we identified essential genes required for growth in rich medium un-der aerobic and microoxic conditions as well as in a defined minimal medium withcitrate as a sole carbon source. Our analysis suggests that 398 genes are required forautonomous growth in rich medium, a number that represents only around 5% ofthe predicted genes of this bacterium. Five hundred twenty-six genes were requiredto support growth in minimal medium, and 434 genes were essential under mi-crooxic conditions (0.5% O2). A comparison of these data sets identified 339 genesthat represent the minimal set of essential genes required for growth under all con-ditions tested and can be considered the core essential genome ofB. cenocepaciaH111. The majority of essential genes were found to be located on chromosome 1,and few such genes were located on chromosome 2, where most of them wereclustered in one region. This gene cluster is fully conserved in allBurkholderiaspe-cies but is present on chromosome 1 in members of the closely related genusRal-stonia, suggesting that the transfer of these essential genes to chromosome 2 in acommon ancestor contributed toward the separation of the two genera.IMPORTANCETransposon sequencing (Tn-Seq) is a powerful method used to iden-tify genes that are essential for autonomous growth under various conditions. In thisstudy, we have identified a set of “core essential genes” that are required for growthunder multiple conditions, and these genes represent potential antimicrobial targets.We also identified genes specifically required for growth under low-oxygen andnutrient-limited environments. We generated conditional mutants to verify theresults of our Tn-Seq analysis and demonstrate that one of the identified geneswas not essentialper sebut was an artifact of the construction of the mutant li-brary. We also present verified examples of genes that were not truly essentialbut, when inactivated, showed a growth defect. These examples have identifiedso-far-underestimated shortcomings of this powerful method.