Proteomic analysis of many species of fungi, particularly filamentous fungi, is difficult due to the lack of publicly available genome sequence data and the problems associated with cross-species comparisons. Furthermore, the detection of fungal proteins in biological systems where there are a greater number of proteins present from other eukaryote species provides additional challenges. We present an EST-based approach for identifying proteins from a fungal endophyte of temperate grasses and demonstrate that this method is well suited for fungi with minimal sequence data.