Epichloë species are endophytic symbionts of grasses which may differ in the relative importance of their sexual or asexual life cycles. Sexual reproduction of the fungus by stroma-formation prevents host flowering (choke) and thus is highly antagonistic, whereas asexual reproduction through clonal propagation in host seeds does not affect host fitness. Stroma-forming Epichloë endophytes from Bromus erectus and non-stromal strains from B. benekenii and B. ramosus were recognized as a distinct mating population (MP) based on complete sexual compatibility among strains and intersterility between other MPs established by mating tests. This biological species represents the only documented case of highly antagonistic strains interfertile with highly mutualistic strains. A second distinct MP of Epichloë was evident on Brachypodium sylvaticum including both stroma-forming and non-stromal isolates. These two MPs were further characterized by distinct morphologies of fruiting structures, allozyme divergence, β-tubulin gene phylogeny, and host preferences, and were described as new species: E. bromicola associated with Bromus spp. and E. sylvatica with Bp. sylvaticum. Additional mating tests among Epichloë from several, previously unexamined, hosts including Brachypodium pinnatum, Calamagrostis villosa, Festuca spp., Phleum pratense, and Poa spp., expanded the known host ranges of three other European species, E. typhina, E. festucae and E. baconii. Genetic variability of all five European species and gene diversity of host subpopulations were analysed based on allozyme data from a total of 497 Epichloë isolates. Average gene diversity (Hs) within MPs ranged from 0·09 to 0·36 with E. typhina being the most diverse, and GST values, a measure for between subpopulation differentiation, ranged from 0·73 to 0·90 indicating that genetic isolation of endophytes on many host grasses is likely.