Diphtheria is a respiratory disease caused byCorynebacterium diphtheriae. While the toxin-based vaccine has helped control outbreaks of the disease since the mid-20thcentury there has been an increase in cases in recent years, including systemic infections caused by non-toxigenicC.diphtheriaestrains. Here we describe the first study of gene essentiality inC.diphtheriae, providing the most-denseTransposonDirectedInsertionSequencing (TraDIS) library in the phylum Actinobacteriota. This high-density library has allowed the identification of conserved genes across the genus and phylum with essential function and enabled the elucidation of essential domains within the resulting proteins including those involved in cell envelope biogenesis. Validation of these data through protein mass spectrometry identified hypothetical and uncharacterized proteins in the proteome which are also represented in the vaccine. These data are an important benchmark and useful resource for theCorynebacterium,Mycobacterium,NocardiaandRhodococcusresearch community. It enables the identification of novel antimicrobial and vaccine targets and provides a basis for future studies of Actinobacterial biology.