Background Tall fescue (Festuca arundinacea Schreb., Lolium arundinaceum Schreb. Darbysh) and perennial ryegrass (Lolium perenne) are important cool‐season forage and amenity grasses that have a mutualistic association with an endophytic fungus. Endophytes confer insect and drought resistance to plants but can produce mammalian toxins. Novel endophytes that do not produce mammalian toxins have been introduced to elite cultivars for commercial production. Seed companies need to maintain adequate levels of novel endophytes within the elite forage cultivars. Endophyte detection is performed using immunochemical and molecular techniques because of their speed and reliability. Early detection in seedlings is essential to evaluate the viability of the endophyte within seed lots. Methods This research aimed to identify the earliest growth stage in which immunochemical and molecular methods can detect viable endophyte in seedlings of tall fescue cultivars BarOptima (e34), Texoma MaxQII (584), and Jesup MaxQ (542), as well as the perennial ryegrass cultivar Remington (NEA2). Results Immunochemical testing detected endophytes in seedlings 14 days after germination (DAG), but the detection rate increased until 42 DAG in some cultivars tested. The molecular marker Tef1exon detected endophytes at a lower rate than the immunochemical method at 28–42 DAG. However, there was insufficient DNA to detect endophytes in 14 DAG seedlings using markers. Conclusions We conclude that the most accurate detection of viable endophytes in seedlings was 42 DAG, at which sufficient and consistent endophyte colonization occurred. Many forage grasses have a beneficial fungus living inside them. The presence of this fungus is a necessity to many producers. Knowing when and how to test for it has been a problem in the past. Here, we compare different methods of fungal testing and different time points that will help producers make testing decisions.