Annual ryegrass toxicity (ARGT) is responsible for significant stock losses in South Australia and Western Australia. The toxicity is caused by corynetoxins produced by the bacterium Rathayibacter toxicus (with the possible involvement of a bacteriophage), which infects annual ryegrass (Lolium rigidum). Polymerase chain reaction (PCR)-based assays, compatible with an existing enzyme-linked immunosorbent assay for the corynetoxins, have been developed and used to screen L. rigidum for both the presence of R. toxicus and for the bacteriophage isolate NCPPB 3778. The results from analysing bacterially infected galls from toxic grain screenings showed a positive correlation between the presence of the bacterium and corynetoxins but not with the bacteriophage. Analysis of pasture-derived samples of annual ryegrass showed about a 50% correlation of corynetoxins with bacterial presence and about a 5% correlation of phage with the presence of the bacterium. These observations support the potential application of the PCR-based assays in providing a useful, complementary tool in the assessment of the likelihood of pasture and feed to cause ARGT and to enable a better understanding of the complex aetiology of ARGT.