Of 683 Botrytis cinerea isolates collected from a fungicide-trial vineyard, 31 were classified as putatively resistant to fenhexamid (50% effective concentration [EC50] ≥ 0.1 μg/ml). For the resistant isolates that survived and sporulated in culture, colony expansion and conidial germination frequency was significantly reduced relative to the mean of 30 representative baseline isolates (EC50 = 0.03 μg/ml). Grape berries were inoculated with four isolates representing a range of fenhexamid sensitivities and treated preventively or curatively with fenhexamid concentrations (150 to 600 mg/liter) representing 25 to 100% of the recommended rate. All treatments significantly delayed disease onset and progress caused by isolates with EC50 values of 0.03 and 0.15 μg/ml but provided little to no control of isolates with EC50 values of 0.32 and 62.5 μg/ml. The latter isolate exhibited a previously unreported F427V mutation of ERG27, an enzyme of ergosterol biosynthesis. In a duplex quantitative polymerase chain reaction test, the ratio of pathogen/host DNA increased significantly for 14 days after inoculation of untreated berries with a baseline isolate but declined slightly in berries treated with fenhexamid at 600 mg/liter 1 day post inoculation. In the vineyard, disease control was affected by the number and rate of fenhexamid applications but B. cinerea isolates with EC50 ≥ 0.1 μg/ml were not preferentially selected.