Rathayibacter toxicus, an APHIS-listed Select Agent, produces a neurotoxigenic glycolipid similar to tunicamycin. Endemic to Australia, R. toxicus is obligately vectored by Anguinid seed gall nematodes to the developing seedheads of forage grasses, where tunicamycin is synthesized and subsequently consumed by foraging livestock. This results in the only known naturally occurring tunicamycin-associated disease, annual ryegrass toxicity of livestock. To investigate whether a tunicamycin-related biosynthetic gene cluster (TGC) was present in other Rathayibacter, all available genome sequences were analyzed. Novel TGCs were identified in R. agropyri, R. iranicus, and the undescribed South African Rathayibacter species “EV”. The putative TGCs shared conserved tunicamycin-related genes essential for toxin production, but differ in gene order, orientation, and number. All tested R. toxicus, R. agropyri, R. iranicus, and R. EV strains possess a putative TGC. Therefore, all three species appear to be capable of synthesizing tunicamycin-like toxins; although it remains to be determined whether and under what conditions toxin is actually produced. Exploration of tunicamycin sensitivity and genetic variation within the TGC of each species is ongoing. The identification of multiple TGCs highlights the conserved nature of tunicamycin-like antibiotics within the Rathayibacter genus.