Determining fungal endophyte presence in seeds non-destructively allows seed and endophyte to remain viable for further experimentation or for use in breeding, genetics or sales. Two methodologies were investigated for endophyte classification and both were successful in segregating endophyte-infected from uninfected annual ryegrass (Lolium rigidum) seeds. Segregation of annual ryegrass seeds was achieved by excising and retaining the embryo section (including some endosperm), and evaluating endophyte presence in the other section of the corresponding seed. Evaluation and segregation of excised seed was completed using either standard microscopy techniques or a commercial immunoblot kit. Both segregation techniques employed were successful in identifying endophyte infection prior to germination. Biomass production in annual ryegrass plants was unaffected by seed excision, indicating the feasibility of the segregation for plant production experiments. Germination was unaffected, although histochemical analysis revealed that the proportion of viable seeds that remained ungerminated at day 21 was higher amongst intact seeds (P < 0.05) compared with excised seeds. The use of the microscopy or immunoblot techniques presented here are viable ways to segregate endophyte-infected from uninfected seeds for future use in experimental or breeding programmes.