Speciation in sexually reproducing organisms hinges on reproductive barriers that reduce gene flow between species or preclude the formation of hybrids. Here, we studied potential reproductive barriers in four members of the Epichloe typhina (Ascomycota, Clavicipitaceae) complex, i.e. Epichloe typhina infecting Dactylis glomerata, E. typhina subsp. clarkii infecting Holcus lanatus, E. typhina subsp. poae infecting Poa nemoralis and E. typhina infecting P. trivialis. Reciprocal inoculation tests showed that these endophytes are host-specific. This suggests that reproductive isolation among Epichloe strains may be the result of specialization to one host, on which mating between different individuals occurs. Furthermore, significantly lower infection frequencies of F1 progeny from crosses between host-strains compared to parental strains and within host-strain progeny suggest that host-dependent effects upon hybrid fitness exist, which would conform to an extrinsic postzygotic isolation barrier. Our results may explain, why members of the E. typhina complex remain genetically differentiated in natural populations. (C) 2015 Elsevier Ltd and The British Mycological Society. All rights reserved.