Lolium rigidum (annual ryegrass), a widespread weed of southern Australian cereal crops, has frequently evolved herbicide resistance. It is commonly found associated with the mutualistic, endophytic fungus, Neotyphodium occultans. The objectives of this study were as follows: (i) determine the occurrence of N. occultans in L. rigidum populations across southern Australia, (ii) evaluate potential associations between infection frequency and herbicide resistance, climatic and topographic variables and (iii) examine genetic diversity of N. occultans from this national collection of L. rigidum using simple sequence repeat markers. By microscopic assessment of seed, we found all seed lots were infected with N. occultans, with highly variable frequencies, ranging from 4% to 98%. Frequencies were higher in New South Wales, South Australia and Victoria (54'62%) than in Western Australia (43%) or Tasmania (17%). A negative association existed between elevation and mean rainfall, and infection frequency. Significant interactions were as follows: mean rainfall (growing season) and state and mean rainfall (growing season) and elevation. Significant associations were found between infection frequency and herbicide mode of action. Infection was lower in seed lots resistant to group B (sulfonylurea and imidazolinone) and higher in seed lots resistant to group D (dinitroaniline) herbicides. Higher infection frequencies were observed in seed lots from earlier years, with the lowest in seed lots recently collected. No significant associations were found with long-term mean temperature or rainfall. Little genetic diversity was observed for N. occultans endophyte in Australian plants. These results provide a benchmark of the relationship of N. occultans with L. rigidum and suggest further research on the role this endophyte has in determining the fitness of L. rigidum under varying conditions of herbicide resistance, rainfall and elevation that may aid in themanagement of this grass.