Klebsiella pneumoniae is a key nosocomial pathogen associated with increasing resistance to first-line antibiotics. Therefore, the identification of novel targets is required for the treatment of infections caused by K. pneumoniae. Genes that are essential for the survival of a pathogen but not a commensal organism might represent good targets. We have used Transposon Directed Insertion-site Sequencing (TraDIS) to generate a highly-saturated mutant library of K. pneumoniae ECL8. The library consists of 476,249 unique transposon insertion sites which represents an average insertion every 10 base pairs throughout the genome. To define whether a gene is essential, the number of transposon insertions normalised for coding sequence (CDS) length was calculated and denoted the insertion index score (IIS). The genome-wide IIS distribution was bimodal. For a given insertion index score, the probability of the gene belonging to the essential or non-essential mode was calculated, and the ratio of these values was represented as a log likelihood score. A gene was classified as essential if its log likelihood score was less than log2 12 and was therefore 12 times more likely to belong to the essential mode than the non-essential mode. A total of 376 K. pneumoniae genes satisfied these stringent criteria and were therefore identified as essential. Analysis of the clusters of orthologous groups (COGs) of the essential genes showed that gene products related to translation, cell wall biogenesis and coenzyme metabolism were enriched. Comparison between the essential genes of E. coli K-12 and K. pneumoniae has revealed key differences between these two organisms. E. coli and K. pneumoniae share 272 essential genes, however we have identified 103 genes that are essential for K. pneumoniae only. These 103 genes represent potential novel candidates for the targeted treatment of K. pneumoniae infections.