Gray mold is a devastating disease on strawberry, and may be caused by several species of Botrytis. The goal of this study was to better understand and characterize the species of Botrytis with reduced sensitivity to the fungicide Polyoxin D, particularly Botrytis fragariae. In total, 78 Botrytis isolates of unknown species that were sensitive (28 isolates; S), moderately sensitive (22 isolates; MS), or reduced sensitive (28 isolates; RS) to Polyoxin-D were collected from commercial strawberry fields of five states in the United States, identified to the species level, and characterized. The majority (75%) of S isolates were Botrytis cinerea and the majority (79%) of RS isolates were the recently described species B. fragariae, indicating an innate ability of B. fragariae to tolerate Polyoxin-D. B. fragariae produced fluffy, white mycelium and was less likely to sporulate on potato dextrose agar than B. cinerea. Isolates from a commercial field recovered from blossoms in early spring were all B. fragariae, those from leaves of the same plants in late spring were a mixture of B. fragariae and B. cinerea, and those from fruit in early summer were all B. cinerea, indicating that B. fragariae may preferentially colonize blossom tissue. A polymerase chain reaction-based assay was developed based on NEP2 sequence variability to distinguish B. fragariae from other Botrytis spp. that have been reported on strawberry, including B. cinerea, B. mali, B. caroliniana, and B. ricini. None of the isolates collected from Canada, California, or North Carolina nurseries were B. fragariae, indicating that the newly described species may not exist or not be widely distributed in planting stock.