Widespread infection of Epichloë occultans in annual ryegrass in Australia suggests that infection provides its weedy host, Lolium rigidum, some ecological advantage. Initial studies determined the distribution and profiles of known Epichloë alkaloids (epoxy-janthitrems, ergovaline, lolines, lolitrem B, and peramine) in plant extracts using a combination of GC-FID and HPLC techniques utilizing a single accession of Australian L. rigidum. However, the lolines N-acetylnorloline (NANL) and N-formylloline (NFL) were the only alkaloids detected and were highly concentrated in the immature inflorescences of mature plants. Additional glasshouse studies subjected a wide range of Australian L. rigidum haplotypes and international annual Lolium accessions to a suite of analyses to determine alkaloid levels and profiles. Again, NFL and NANL were the key lolines produced, with NFL consistently predominating. Considerable variation in alkaloid production was found both within and between biotypes and accessions evaluated under identical conditions, at the same maturation stage and on the same tissue type. The pyrrolopyrazine alkaloid peramine was also present in 8 out of 17 Australian biotypes of L. rigidum and 7 out of 33 international accessions infected with Epichloë spp.; the highest peramine concentrations were observed in seed extracts from L. rigidum collected from Australia. This study represents the first report of alkaloids from a geographically diverse collection of annual ryegrass germplasm infected with Epichloë spp. when grown under identical controlled conditions.