Pastures of tall fescue (Festuca arundinacea Schreb.) infected with the endophytic fungus Acremonium coenophialum Morgan‐Jones and Gams have been associated with livestock disorders known collectively as fescue toxicosis. Destruction of infected (E+) fescue pastures and reestablishment with endophyte free (E−) seed is beneficial for eliminating fescue toxicosis. However, since management decisions must be based on knowledge of A. coenophialum incidence, sampling of pastures should provide information about fungal incidence with accuracy. Two experiments were conducted to compare sampling methods. In the fist experiment, eight 4‐ha, 2‐yr‐old pastures established with seed mixtures ranging from near 0 to more than 70% E+ were sampled. A transect method and a stratified random sample based on area at an intensity of 23 tillers ha−1 were used. In the second experiment, four 20‐yr‐old, 2‐ha E+ pastures were sampled at monthly intervals for 2 yr. Stratified random sampling at 41 samples ha−1 and simulated transects using the same data were used. Samples were assessed for E+ status using Protein A enzyme‐linked immunosorbent assay (PAS‐ELISA). In the first study, observed E+ incidence in two pastures increased from 15 or 30% to about 60%, respectively, during the 2‐yr. Reasons for this increase are not known. The other pastures, established with 0,45, 60, and 75% E+ seed had only small increases in E+ incidence. Significant E+ incidence could be detected in the second study at any time during the year. Six to eight samples ha−1 were adequate for characterizing A. coenophialum status. Transect and stratified random samplings gave similar E+ estimates.