Analysis of Amplified Fragment Length Polymorphisms (AFLP) in a sample population of Epichloë and Neotyphodium was conducted to evaluate the usefulness of this technique in estimating phylogenetic relationships among these fungi. A total of 963 unique restriction fragments were identified with 13 selective primer combinations. Neighbour-joining and parsimony analysis produced phylogenetic trees with high bootstrap support. All Epichloë festucae isolates were monophyletic, and clades of E. festucae were identified that correspond to the host species from which they were isolated. These groups may indicate the importance of coevolution in the development of the fine fescue-E. festucae symbiosis. The grouping of E. festucae isolates from hard fescue and Chewings fescue into one clade conflicts with the current taxonomy of these host grasses. Sequencing of ribosomal DNA spacer regions ITS1 and ITS2 was conducted for comparisons to the phylogenetic relationships estimated by AFLP. Using the maximum likelihood test, a significant difference was detected between AFLP and rDNA trees due to discrepancies in the association of anamorphs (Neotyphodium) and teleomorphs (Epichloë). This discrepancy may be due to some mechanism of genetic recombination, such as hybridization or parasexual recombination, that occurs in Neotyphodium populations. It is concluded that AFLP markers are useful for producing meaningful phylogenies among a diverse group of fungi and for identifying groups of closely related individuals that are genetically and biologically similar. Further investigation is needed to determine how AFLP markers change over short periods of time in populations and over evolutionary time.