Clavibacter michiganensis causes bacterial wilt and canker in a variety of crops. It is subdivided into five subspecies according to host plant specificity. In recent years, several virulence factors have been identified in C. michiganensis subspecies, however, most of those are identified in C. michiganensis subsp. michiganensis (Cmm) and C. michiganensis subsp. sepedonicus (Cms). None of the identified virulence factors have closely related genes in C. michiganensis subsp. nebraskensis (Cmn), which is a responsible for wilt and blight of maize, and we lack an understanding of molecular mechanisms in the Cmn interaction with maize. The development of molecular resources and techniques in Cmn is a required step towards understanding Cmn pathogenesis. Here, a Cmn transposon mutant library was constructed and screened to identify mutants with reduced or no virulence. Targeted mutagenesis was also performed to knock out virulence factor candidate genes identified from comparative genomics between virulent and non-virulent Cmn isolates.