Pasteurella multocida is a Gram-negative coccobacillus and the causative agent of various animal diseases including avian cholera, haemorrhagic septicaemia in ungulates, shipping fever in cattle and atrophic rhinitis. Collectively, these disease syndromes have a large economic impact on production animal industries worldwide. Transposon-directed insertion site sequencing (TraDIS) can comprehensively identify genes essential for the survival of a bacterium in any specific niche. The first step in TraDIS is the production of a very large (near saturation) library of single-insertion transposon mutants. The mutant library is then grown in the particular condition of interest; mutants with an insertion in an essential gene or region will fail to grow and will be lost from the population. The surviving bacteria are recovered, and all transposon insertion sites are identified by high-throughput sequencing. Genes/regions that lack transposon insertions are assumed to be essential for growth in the condition in which the bacteria were grown. In order to identify essential P. multocida genes we used the Himar1 transposon to produce a high density mutant library in the fowl cholera-causing isolate P. multocida strain VP161. The mutant library contained >70,000 mutants, which corresponds to a unique transposon insertion every 28 nucleotides. Following growth in rich medium (BHI) we identified 17 tRNAs and approximately 450 genes that are known to be essential in other bacterial species, including 49 50s and 30s ribosomal protein genes. Importantly, we also identified 47 open reading frames in P. multocida strain VP161 that have not been previously identified as essential in any other bacterial species; 27 of these encode hypothetical proteins. Investigation of these essential genes will give novel insight into the growth requirements of P. multocida. TraDIS analyses of P. multocida mutant libraries grown in conditions that simulate host niches, including iron-depleted medium and 90% serum, are ongoing.