Charles Sturt University has operated a commercial herbicide resistance testing service since 1991, following a random survey of the South West Slopes region of New South Wales that identified significant incidence of herbicide resistance in annual ryegrass (Lolium rigidum Gaud.). Other surveys of cropping regions of southern Australia conducted at that time also found a significant incidence of resistance. In the subsequent 25-year period, the testing service has received samples from the majority of the southern Australian cropping belt. Overall, 80% of samples tested were resistant to acetyl-CoA carboxylase (ACCase) inhibiting aryloxyphenoxypropionate and phenylpyrazole herbicides, 56% to acetolactate synthase (ALS) inhibiting herbicides, and 24% to ACCase-inhibiting cyclohexanedione herbicides. The incidences of resistance to inhibitors of photosynthesis at PSII, tubulin-formation inhibitors, and 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase inhibiting herbicides have remained <10% of samples tested. The relationships between many herbicide groups and subgroups are discussed, as is the variability in resistance incidence and the forms of cross or multiple resistance for each state. This paper builds on an earlier publication of 14 years of testing history. At >5000 samples, the size and geographical spread of this dataset allows for valuable analyses of the relationships present in herbicide-resistant populations of annual ryegrass.